Immunomagnetic Columns Research Articles

MIR4435-2HG as a possible novel predictive biomarker of chemotherapy response and death in pediatric B-cell ALL.

Introduction: Although B-cell acute lymphoblastic leukemia (B-cell ALL) survival rates have improved in recent years, Hispanic children continue to have poorer survival rates. There are few tools available to identify at the time of diagnosis whether the patient will respond to induction therapy. Our goal was to identify predictive biomarkers of treatment response, which could also serve as prognostic biomarkers of death, by identifying methylated and differentially expressed genes between patients with positive minimal residual disease (MRD+) and negative minimal residual disease (MRD-). Methods: DNA and RNA were extracted from tumor blasts separated by immunomagnetic columns. Illumina MethlationEPIC and mRNA sequencing assays were performed on 13 bone marrows from Hispanic children with B-cell ALL. Partek Flow was used for transcript mapping and quantification, followed by differential expression analysis using DEseq2. DNA methylation analyses were performed with Partek Genomic Suite and Genome Studio. Gene expression and differential methylation were compared between patients with MRD-/- and MRD+/+ at the end of induction chemotherapy. Overexpressed and hypomethylated genes were selected and validated by RT-qPCR in samples of an independent validation cohort. The predictive ability of the genes was assessed by logistic regression. Survival and Cox regression analyses were performed to determine the association of genes with death. Results: DAPK1, BOC, CNKSR3, MIR4435-2HG, CTHRC1, NPDC1, SLC45A3, ITGA6, and ASCL2 were overexpressed and hypomethylated in MRD+/+ patients. Overexpression was also validated by RT-qPCR. DAPK1, BOC, ASCL2, and CNKSR3 can predict refractoriness, but MIR4435-2HG is the best predictor. Additionally, higher expression of MIR4435-2HG increases the probability of non-response, death, and the risk of death. Finally, MIR4435-2HG overexpression, together with MRD+, are associated with poorer survival, and together with overexpression of DAPK1 and ASCL2, it could improve the risk classification of patients with normal karyotype. Conclusion: MIR4435-2HG is a potential predictive biomarker of treatment response and death in children with B-cell ALL.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

The LSD1 Inhibitor RN-1 Increases γ-Globin Expression in Baboons By Targeting an Early Event Responsible for γ-Globin Repression

The HbF to HbA developmental globin switch is recapitulated during adult erythroid differentiation and involves the acquisition of repressive epigenetic marks at the γ-globin promoter catalyzed by “druggable” enzymes such as histone deacetylases (HDACs), DNA methyltransferase (DNMT1), LSD1 (KDM1A), and G9A (EHMT2) that are functional components of multiprotein co-repressors recruited to the γ-globin gene promoter by the trans-acting repressors TR2/TR4, BCL11A, and ZBTB7A. A logical approach to increase HbF that has been successfully pursued by our laboratory is to intervene with the epigenetic repression mechanism that executes the switch from HbF to HbA using pharmacological inhibitors of these enzymes. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates.Our laboratory has developed and utilized an in vivo baboon model for over thirty years to investigate globin gene regulation and the ability of pharmacological inhibitors of enzymes that catalyze repressive epigenetic modifications to increase HbF, generating results with DNMT inhibitors that were extended and confirmed in a number of clinical studies in patients with SCD and β-thalassemia. Following studies that identified LSD1 as an additional therapeutic target (Shi et al, Nat Med 19:291, 2013), we showed that the LSD1 inhibitor RN-1 dramatically increased HbF, F cells and F retics in baboons (Rivers et al, Haematol 101:698, 2015) and that these effects were sustained upon long-term treatment (>265d; Ibanez et al, Blood 129:260, 2017). ChIP analysis showed increased levels of Histone H3 di and tri-methyl K4 at the γ-globin gene, consistent with LSD1 inhibition. This current investigation seeks to identify the stage in the erythroid differentiation pathway targeted by LSD1 inhibitors to increase HbF and also to characterize additional effects of these drugs on erythroid differentiation that potentially impact mechanism of action. Subpopulations of bone marrow (BM) cells highly enriched in BFUe (CD105+CD34+CD117+bRBC-), CFUe (CD105+CD34+CD117+bRBC+), proerythroblasts (CD105+CD34-CD117+bRBC+) were purified from RN-1 treated (0.25mg/kg/d; 3d) and untreated baboons by immunomagnetic column separation combined with FACS. RT-PCR analysis of γ-globin expression within purified BM erythroid subpopulations showed that relative levels of γ-globin mRNA (γ/γ+β) in untreated baboons were 4 fold higher in BFUe than in CFUe and 11 fold higher in BFUe than in proerythroblasts consistent with repression of the γ-globin gene during the BFUe to CFUe transition. RN-1 treatment increased relative γ-globin expression (γ/γ+β) 4 fold in BFUe, 25 fold in CFUe, and >100 fold in proerythroblasts compared to untreated controls and thus partially prevented repression of the γ-globin expression at early stages of differentiation. Additional effects of the LSD1 inhibitor RN-1 on erythroid differentiation were measured by flow cytometry in baboon BM aspirates following RN-1 administration. RN-1 treatment (0.25mg/kg/d; 3d) increased the proportion of CD105+CD117+bRBC+BM cells (proerythroblasts) (24.2% pre-treatment; 48.4% post-treatment; p<0.02) and decreased the proportion of CD105+CD117-bRBC+ terminal erythroid BM cells (51.1% pre-treatment; 29.1% post-treatment; p<0.02). RNAseq analysis of CD105+CD34-CD117+bRBC+ FACS-purified BM cells (proerythroblasts) of untreated control (n=2) and RN-1 treated baboons (0.25mg/kg/d; 3d; n=4; 2 treated for 3d; 2 treated >265d) identified a common set of 41 genes whose expression was increased while the expression of 6 genes was decreased. RT-PCR assays confirmed increased expression of γ-globin, GATA-2, GFi-1B, DAP, IFI6, and Lyn in proerythroblasts of RN-1-treated baboons. We conclude that the LSD1 inhibitor RN-1 administered to baboons 1) restrains erythroid differentiation by modulating the expression of genes such as GATA-1 and GFi-1B that regulate erythroid differentiation, and 2) targets an early event responsible for γ-globin repression to increase γ-globin expression. DisclosuresSaunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Lavelle:Global Blood therapeutics: Research Funding.

Impact of Busulphan Exposure on Transplant Outcomes for Patients with Advanced Myelodysplastic Syndromes Undergoing CD34 Selected Allogeneic Hematopoietic Cell Transplantation

CD34 selected stem cell transplants (SCT) have shown similar survival rates as unmodified SCT, with lower incidence of acute and chronic graft-versus-host disease (GVHD). A common conditioning regimen for patients with advanced myelodysplastic syndrome (MDS) undergoing CD34 selected SCT is a combination of busulphan, melphalan, fludarabine, and anti- thymocyte globulin (ATG). The ideal area under the curve (AUC) and overall dose intensity of busulphan is unknown in these pts.We aimed to study the relationship between busulfan AUCs and transplant outcomes in this pt population.This retrospective analysis included 68 pts with advanced MDS (RAEB-I and higher) who underwent CD34 selected SCT between 2000-2012. Median age was 58.05 yrs (26-73). There were 36 women (52.9%). HCT CI was ³ 3 in 37 pts (54.4%). MDS subtype at diagnosis by WHO criteria was: RA/RCMD-19 (all progressed to RAEB/AML), RAEB-I 20 &RAEB II 29; and by IPSS-R criteria in 65 pts: very low risk-4, low risk-9, intermediate risk-14, high risk-20 & very high risk-18. All pts were conditioned with busulphan IV 0.8 mg/kg dose, melphalan 140 mg/m2, fludarabine 125 mg/m2 & ATG . Thirty-nine pts received 10 doses of busulphan and 29 pts 12 doses. The dose was increased to reduce the relapse rate. Pharmakokinetic studies were done after first busulphan's dose and based on results adjustments were made to achieve a target dose of 1025-1315µM x min. G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Donors were HLA matched, 48 (18 related & 30 unrelated) or mismatched unrelated, 20.The median first-dose busulphan AUC was 1,206 (723-2,180). In 27 pts (39.7%) this was within target range of 1,025-1,315 with 16 pts (23.5%) below and 25 (36.7%) above. The median total busulphan exposure for all pts was 12,822 (9,513-15,754), with 1st quartile range of 9,513-12,181 and 4th quartile range of 13,664-15,754. The median total exposure in the 10 doses group (group 10) was 12,520 (9,513-15,754) and in the 12 doses group (group12) 13,420 (9,860-15493). The 2-years overall survival (OS) was 61.5% (CI 44.5-74.7) in group 10 and 55.2%(35.6-71) in group 12 and 2-years relapse free survival (RFS) was 56.4% (39.6-70.2) in group 10 and 51.7% (32.5-67.9) in group 12. The 2-years cumulative incidence of relapse was 15.4% (CI 3.9-26.9) in group 10 and 13.8% (CI 0.9-26.6) in group 12. The non-relapse (NRM) mortality at 2 years was 28.2% (13.8-42.6) in group 10 and 34.5% (16.7-52.2) in group 12. aGVHD grade II-IV was 12.8% (2.2-23.5) in group 10 vs 23.1% (6.5-39.7%) in group 12. None of these differences were statistically significant.Further analysis by quartiles showed no differences in OS, RFS, relapse and NRM between the 4 groups. However, grade II-IV acute GVHD was significantly higher in the 4th quartile exposure, 35.6% (8.9-62.3) vs 5.9% (0-17.5), 11.8% (0-27.6) and 17.6% (0-36.4) in the 1st, 2nd and 3rd quartiles (p=0.046) (table 1).In thishomogenous cohort of pts, total busulphan exposure was only found to be associated with grade II-IV aGVHD, with higher incidence seen in pts who had exposure >13,664. Donor mismatch status was not associated with higher aGVHD in this cohort and therefore can not explain the increased aGVHD seen with higher busulphan exposure. A trend for better transplant outcomes was seen with total exposure of 12,182- 13,663 (2nd and 3rd quartiles); though the differences were not statistically significant. In CD34 selected allo-HSCT for pts with advanced MDS, the intensity of the conditioning has an impact on transplant outcomes and therefore determining the range of busulphan that offers best survival with minimal GVHD has important clinical implications.Table 1Transplant outcomes (cumulative incidence with 95% CI):Total exposure6-months CI ofaGVHD II-IV2-year CI of Relapse2-year CI NRM2-year CI OS2-year CI RFSQ1: 9,513-12,181, n=175.9 (0-17.5)35.3 (11.6-59)23.5 (2.4-44.7)52.9 (27.6-73)41.1 (18.6-62.6)Q2: 12,181-12,822, n=1711.8 (0-27.6)11.8 (0-27.7)23.5 (2.6-44.4)70.6 (43.1-86.6)64.7(37.7-82.3)Q3: 12,823-13,663, n=1717.6 (0-36.4)5.9 (0-17.5)35.3 (11.7-58.9)58.8 (32.5-77.8)58.5 (32.5-77.8)Q4: 13,664-15,754 n=1735.6 (8.9-62.3)5.9 (0-17.4)41.2 (16.8-65.6)52.9 (27.6-73)52.9 (27.6,-73.0)p value*overall: 0.185, Q4 vs others: 0.0460.2210.4090.6630.540 [Display omitted] DisclosuresGiralt:SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding.

Diethylnitrosamine genotoxicity evaluated in sprague dawley rats using Pig‐a mutation and reticulocyte micronucleus assays

Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA-damaging activities are not usually evident in hematopoietic cells because the short-lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood-based endpoints of genotoxicity that have been automated using flow cytometric analysis-frequency of micronucleated reticulocytes and Pig-a mutant phenotype reticulocytes (RET(CD59-) ) and erythrocytes (RBC(CD59-) ). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RET(CD59-) and RBC(CD59-) frequencies were determined on Days 15, 29, and 42. The Pig-a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RET(CD59-) frequencies were significantly elevated in the high dose group on Day 29, and RBC(CD59-) were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig-a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA-damage.

In vitro effects of imatinib on CD34+ cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase

Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34+ cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34+ cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34+ cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase.

Evaluation of Leukemic Stem Cell Persistence in Chronic Myeloid Leukemia (CML) Patients in Complete Molecular Remission Induced by First Line TKI Therapies

AbstractAbstract 3726Recent data showed that BCR-ABL expressing leukemic stem cells (LSCs) persist in vivo in patients in complete molecular remission (CMR) according to European Leukemia Net criteria (Chomel et al, Blood 2011, Chu et al, Blood 2011). It has also been established that, in this category of patients (CMR 4.5– 5), Imatinib Mesylate (IM) discontinuation leads to relapse in the majority of cases (60%), especially during the first 6 months. Although very likely, a formal link has not been established between these two phenomena and, in particular, the prevalence of LSC persistence in a cohort of patients treated with TKI as a first line therapy has not been established. To determine the prevalence of LSC persistence in CMR induced by first line TKI therapies, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL expressing LSCs in marrow samples of 26 patients in CMR for > 2 years. CMR was induced by IM (n=22), Dasatinib (n=3) or Nilotinib (n=1) as a first line treatment. CD34+ cells were isolated from bone marrow aspirates (2–4 ml) using immunomagnetic columns and a clonogenic assay was performed. At week+2, clonogenic progenitors were counted and 20 to 40 individual hematopoietic colonies were plucked from methylcellulose for RNA extraction. In some cases, pooled CFU-Cs were also analyzed (20 pools of 10 hematopoietic colonies). The same CD34+ marrow sample was used to start LTC-IC assays, which were performed in murine MS5 cell feeders. At week+6 the cultures were sacrificed and the number of LTC-IC-derived clonogenic cells was determined. Hematopoietic colonies were plucked as decribed above for CFU-Cs for RNA extraction and BCR-ABL expression was quantified by q-RT-PCR. For 4 patients, the yield of CD34+ cells was not sufficient for stem cell assays (3 pts treated with IM; 1 with Dasatinib). qRT-PCR analyses of CFU-Cs revealed the presence of BCR-ABL expressing cells in 3 patients out of 16 tested so far. In all three patients in CMR induced by IM, this involvement can be estimated to approximately 100–360 leukemic CFU-GM/ml of bone marrow. Concerning LTC-IC assays, 2 patients out of 6 tested so far had evidence of BCR-ABL expressing LTC-IC-derived progenitors, one of these patients having also involvement of the clonogenic compartment. In both patients, there was a significant involvement of LTC-IC-derived progeny estimated to 1000–4000 LTC-IC derived progenitor/ml of bone marrow. In the 4 patients in whom LTC-IC compartment was found to be negative for LSCs, CMR was induced by IM in 3 Pts (IM 400 mg n= 2, IM 800 mg for accelerated phase n=1) and by Dasatinib (100 mg) in 1 Pt. Interestingly, in all q-RT-PCR assays, the amount of BCR-ABL mRNA (evaluated by the BCR-ABL/ABL ratio) in individual CFU-Cs appeared to be low, confirming our previous results (Chomel et al Blood 2012) and suggesting that these cells are not addict to BCR-ABL-TK activity for their survival. In 12 patients, the evaluation of LTC-IC compartment is underway. Overall, These data reveal that in patients in CMR induced by IM as a first line therapy, our strategy could identify two types of stem cell response, with some patients presenting a complete eradication of the leukemic stem cell compartment (within the limits of the sensitivity of the test) and others showing high numbers of BCR-ABL expressing stem cells despite the continuous CMR status on TKI therapy. It remains to be determined if the involvement of LTC-IC-compartment (in 2/6 analyzed so far) by LSC expressing low levels of BCR-ABL can be overcome by long-term TKI therapy in some patients. In addition, it will be of interest to determine if a correlation exists between LSC-positive patients the potential occurence of a molecular relapse upon discontinuation of TKI therapy. From the mechanistic point of view, the persistence of residual marrow LSCs in some CML patients in CMR strongly suggests the presence of active mechanisms of either LSC retention and/or LSC quiescence-promoting interactions within in the hematopoietic niche. Finally, the comparison of the LSC persistence phenomenon in CMR induced by TKI2 versus IM as first line treatment will be of major interest to determine the LSC eradication potential of these drugs. Disclosures:Rea:Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Turhan:Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

A Phase II Trial of Peri-Transplant Palifermin with Busulfan, Melphalan and Fludarabine Followed by T-Cell Depleted Hematopoietic Stem Cell Transplants in Patients with Advanced Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) Evolved From MDS

AbstractAbstract 1935 Introduction:The success of allogeneic HSCT in the treatment of advanced MDS has been limited by high incidence of relapse and transplant-related mortality (TRM). The main complications contributing to TRM are graft-vs-host disease (GvHD) and infections. Although T cell depletion has successfully reduced the incidence of GvHD, slow immune reconstitution and high rate of infections have compromised its full potential. T cell reconstitution after transplant requires a functional thymic epithelium. Transplant conditioning regimens damage the thymus and impair the production de novo of T cells. Keratinocyte growth factor (KGF) has an important role in healing the epithelium after injury. Palifermin, a human recombinant form of KGF, decreases the incidence and duration of mucositis after total body irradiation and has been approved by the FDA for this purpose in autologous and allogeneic HSCT. In murine models, KGF given before allogeneic transplant has a protective effect on the thymus and accelerates T cell reconstitution.The aim of this study was to assess whether administration of palifermin peri-transplant decreases TRM and improves the overall and disease-free survival (OS and DFS). This study was designed to reduce one- year TRM from 30% and 35% to 10% in HLA matched and mismatched arms, respectively. Patients:Between 11/2009–05/2012, 42 patients (advanced MDS: 26 or AML evolved from MDS: 16) enrolled on this trial. At transplant, 23 were in CR1, 3 were in second refractory cytopenia phase, and 16 had limited disease (<5% circulating blasts and <9% marrow blasts). The median age was 57.5 years (1–65), with 22 males and 20 females. Conditioning consisted of busulfan (12 doses over three days of 0.8 mg/kg IV for patients > 4 years old or 1.0 mg/kg IV for patients < 4 years old), melphalan (70 mg/m2 IV × 2 days), fludarabine (25 mg/m2 IV × 5 days), and rabbit ATG pre-transplant (2 doses and 3 doses for HLA matched and mismatched recipients, respectively). Palifermin was given according to the approved dose for mucositis prevention: 60 mcg/kg/day IV for 3 consecutive days before the preparative regimen, and 3 doses post transplant (day 0, +24hours, +48hours). Donors were HLA matched (31; 13 related and 18 unrelated) or unrelated mismatched (11). G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Results:All 42 patients engrafted, 1 patient developed secondary graft failure. The cumulative incidence of grade III-IV aGvHD at day +100 and 1 year were 4.8% and 10.5%, respectively.The latter increase was due to late-onset acute GvHD. Only 1 of 33 patients at risk developed moderate chronic GvHD. The 2-year OS and DFS were 77% and 65%, respectively, and similar in the two arms (HLA matched and mismatched). The CI of relapse at 1-year was 12 % (similar in the two arms). The 1-year TRM was 20%; 18.4% in the HLA matched group (accrual met) and 22% in the mismatched arm (accrual ongoing). Causes of death were: infections (N=5; 3 HLA matched, 2 HLA mismatched), regimen related toxicity (N=2, liver VOD) and relapse (N=1). The frequency of infections was similar to historical controls; at 3 months post transplant, 30% of patients developed active CMV, EBV, or adenovirus infections, at 6 months 16.6%, and at 12 months 7.1%. The 6-month CI of CMV viremia in CMV seropositive patients was 75% and the 6-month CI of EBV viremia was 31%. Immune reconstitution as measured by CD4 count was slow, similar to historical controls, with median absolute CD4 count at 3 months of 77 cells/μl and 200 cells/μl by 1 year posttransplant. Other parameters to assess safety of this regimen, namely duration of narcotics use and days on TPN to measure severity of mucositis were similar to historical controls (same preparative regimen without palifermin). Conclusion:In this ongoing phase II trial the addition of peri transplant palifermin to a chemotherapy only myeloablative conditioning regimen in recipients of TCD HSCT for advanced MDS decreased the TRM, although did not meet the primary objective of this study (reduction to 10%). Also, there was no reduction in the incidence of viral infections and no improvement in immune reconstitution. The reduction in TRM resulted from early detection of infection and improved treatment options. [Display omitted] Disclosures:Perales:SOBI Biovitrum, pharma company: SOBI Biovitrum, pharma company Other. Goldberg:SOBI Biovitrum: Research Funding.

Poor Graft Function in Recipients of T Cell Depleted (TCD) Allogeneic Hematopoietic Stem Cell Transplants (HSCT) Is Mostly Related to Viral Infections and Anti-Viral Therapy.

AbstractAbstract 3147 Introduction:Poor graft function (PGF) without immune rejection, defined as persistent cytopenias with hypocellular marrow and full donor myeloid chimerism, can be a life-threatening complication after allogeneic HSCT. It is commonly caused by viral infectious, myelosuppressive drugs like antivirals, and graft-vs-host disease (GvHD). Treatment options include supportive therapy with transfusions and growth factors and in severe cases administration of additional hematopoietic stem cells (HSCs) from the same donor without conditioning (stem cell boost). The incidence, natural history, and the indications for stem cell boost therapy are not well defined. Aims:To assess the incidence, etiologies, and indications for stem cell boost for PGF in a homogeneous group of patients with advanced MDS and AML who underwent TCD HSCT from matched or mismatched related or unrelated donors after conditioning with the same myeloablative regimen. Patients and methods:Poor graft function was defined as persistent neutropenia (ANC <1,000 μL and G-CSF administration x3 in 30 days), thrombocytopenia (platelets <50,000 μL or platelets transfusion × 4 in 30 days), and/or hemoglobin <8 g//dL after engraftment with hypocellular BM and full donor myeloid chimerism. Severe PGF was defined as ANC <500 μL, red cell transfusion-dependent anemia with reticulocytopenia of < 20,000 μL, and platelets <20,000 μL. The patient population in which this study was done included 42 patients enrolled between 09/2009 and 05/2012 in a phase 2 trial of palifermin peri-transplant to reduce transplant-related mortality. The median age was 57.5 years (1–65). All patients received the same myeloablative conditioning regimen with busulfan, melphalan, fludarabine, rabbit ATG and palifermin peri-transplant. G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Donors were HLA matched (31; 13 related and 18 unrelated) or mismatched unrelated (11). Chimerism was determined in bone marrow as well as neutrophils, B cells, and T cells by short tandem repeat analysis on DNA extracted from bone marrow and peripheral blood cell subsets. Results:Forty-one patients were evaluable for this analysis; 1 patient was not included as he rejected the allograft shortly after engraftment. There were 8 cases of PGF with a cumulative incidence (CI) at 1 year of 18% (13% HLA matched, 33% HLA mismatch). The etiology was infection in 7 cases, and unknown in the 8th case. This patient presented with presumed autoimmune anemia and thrombocytopenia associated with a hypercellular marrow and did not respond to multiple lines of therapies. Her marrow became later hypocellular and met the criteria for PGF. None of the PGF cases in this series was associated with GvHD at the time of diagnosis of PGF. The infectious etiologies included: 6 viral infections and 1bacterial sepsis + myelosuppressive drugs. The most common viral etiology associated with PGF was CMV (50%). The 1-year CI of PGF in CMV seropositive patients was 25% and in CMV seronegative patients was 14%. Of note, HHV6 viremia was detected in patients with PGF. HHV6 is not routinely monitored, however, making it difficult to establish a causative role. All patients had moderate PGF at diagnosis and 3 cases had worsening of cytopenias and met the criteria for severe PGF. To date, 3 PGF patients have died from EBV-PTLD, adenovirus infection or GVHD (developed after CMV treatment with liposomal cidofovir), 3 continue to suffer from PGF and 2 patients are alive with recovered good blood counts after eradication of CMV. Of the 3 patients with persistent PGF, one received a TCD boost with no response, and 2 continued to be treated for CMV viremia. A stem cell boost was indicated if pancytopenia persisted despite eradication of cause of the PGF. In this small series, there were not enough events to evaluate association between PGF and CD34 cell dose, CD3 cell dose or day 100 T-cell chimerism. Conclusions:In this homogenous population of patients with MDS who underwent TCD allogeneic HSCT, the incidence of PGF is about 20%. The most common cause was viral infection with predominance of CMV. Therefore, strategies to prevent CMV reactivation in patients undergoing allogeneic HSCT has the potential to reduce the risk of PGF and avoid the need for infusion of additional stem cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Combinatorial Effect of Pharmacological Inhibitors of Lsd-1 and DNMT on γ-Globin Synthesis in Cultured Baboon Bone Marrow Erythroid Progenitors

Abstract 249Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New therapies to increase HbF levels in patients are urgently needed. Previous studies from our laboratory have shown that decitabine, an inhibitor of DNA methyltransferase (DNMT), increases HbF to therapeutic levels in non-human experimental primates and SCD patients, thus demonstrating that drugs targeting repressive epigenetic modifications can be effective HbF-inducing agents. Both DNMT1 and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl- histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298–3311, 2011). Experiments were performed to test the effect of the pharmacological LSD-1 inhibitor, tranylcypromine (TC), alone and in combination with the DNA methyltransferase inhibitor decitabine (DAC), on γ-globin expression. Baboon bone marrow (BM) CD34+ erythroid progenitors were isolated from BM aspirates of normal baboons and grown in a co-culture system with the AFT024 murine fetal liver cell line in Iscove’s media containing 30% fetal bovine serum, 200ng/ml SCF, 2U/ml epo, and 1μM dexamethasone. TC (0.5 to 5.0μM) was added to cultures on d7 and d10, while decitabine (0.1μM) was added on d7. On d14 erythroid cells were purified by immunomagnetic column chromatograpy using a baboon-specific anti-red blood cell antibody (BD Bioscience). Effects on globin expression were measured by biosynthetic radiolabelling of nascent globin chain synthesis in the presence of [3H] leucine followed by HPLC separation of individual globin chains. DNA was isolated and effect of the drugs on DNA methylation of the γ-globin promoter determined by bisulfite sequence analysis. Effects on erythroid differentiation were investigated by examination of Wright’s stained cytospin preparations. In three independent cultures TC induced dose-dependent increases in γ-globin chain synthesis (γ/γ+β). In addition TC and DAC in combination increased γ-globin chain synthesis in an additive manner (see Table).CD34+ cell sourceTC (μM)DAC (μM)γ/γ+βPA 7680000.0600.10.325.000.305.00.10.62PA 7482000.0900.10.352.500.165.000.302.50.10.545.00.10.68Bisulfite sequence analysis showed that increased γ-globin chain synthesis in TC-treated cultures was not associated with increased DNA hypomethylaton of the γ-globin promoter. Examination of Wright’s stained cytospin preparations of d14 cultures showed increased numbers of basophilic and polychromatic erythroblasts in cultures exposed to TC compared to untreated controls suggesting that TC reduced the rate of erythroid differentiation. In contrast, numbers of basophilic and polychromatic cells were reduced and orthochromatic cells were increased in cultures treated with TC and DAC compared to TC alone. We conclude that TC increased γ-globin chain synthesis in a dose-dependent manner in baboon erythroid progenitor cell cultures while TC and DAC, in combination, produced additive effects on γ-globin chain synthesis with minimal effects on erythroid differentiation. These results suggest that combinatorial use of epigenetic modifiers may be superior to single agents and support further studies to determine whether TC and other LSD-1 pharmacological inhibitors can effectively be used in combination with DNMT inhibition to increase HbF levels in vivo. Disclosures:No relevant conflicts of interest to declare.

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig-a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig-a endpoint is suitable for both repeated-dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on-going toxicology studies, and use as a short-term assay that provides efficient screening and mechanistic information in vivo.

Glucocorticoid Receptor Translational Isoforms Contribute to Distinct Glucocorticoid Responses of Neutrophils and Eosinophils

Neutrophils, but not eosinophils, have been suggested to mediate steroid resistance in asthma and COPD, the mechanisms of which are unknown. We determined whether the recently described glucocorticoid receptor (GR) translational isoforms underlie the distinct steroid sensitivity of neutrophils and eosinophils. Neutrophils and eosinophils from deidentified human blood samples were isolated to 97% purity by gradient centrifugation, immunomagnetic column separation, and fluorescence-activated cell sorting. Sensitivity of isolated cells to glucocorticoids was examined by measuring apoptotic markers such as annexin-V and caspase 3 activation. GR translational isoforms were determined using Western blot analyses. The pro-apoptotic GR-A isoform was five-fold higher in eosinophils than in neutrophils, whereas the non-apoptotic GR-D isoform was five-fold higher in neutrophils compared to eosinophils (n=9, Student's t-test, p<0.05). The ratio of GR-A/D in eosinophils was more than ten-fold higher than that in neutrophils (p<0.05). Dexamethasone (1 uM, 16 hr) increased eosinophil apoptosis by almost 50%; in contrast, neutrophil apoptosis decreased by approximately 40% in the presence of dexamethasone (p<0.05 for both). These distinct steroid responses in eosinophils and neutrophils were blocked by RU486 (1 uM, 16 hr), a selective GR antagonist, indicating that selective GR isoforms play a role in the different steroid sensitivities of eosinophils and neutrophils. GRβ, a splice variant of GR previously implicated in steroid resistance, was undetectable in either cell type. Neutrophils and eosinophils have distinct GR translational isoforms. The GR-D isoform in neutrophils likely mediates their resistance to steroids and may underlie steroid-resistant diseases characterized by neutrophil-predominant inflammation.

Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.

Interlaboratory Pig‐a gene mutation assay trial: Studies of 1,3‐propane sultone with immunomagnetic enrichment of mutant erythrocytes

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.

Abstract P6-04-09: Single Cell Whole Genome Amplification for High Density SNP Analysis of Circulating Tumour Cells in Early Breast Cancer

Abstract Background: Despite progressive advances in the fields of radiation and chemotherapy, metastasis remains the leading cause of death in women with recurrent breast cancer. In metastasis, cells disseminate from the primary tumor, and circulate via the vascular system to distant organs, developing tumors at these new sites. Recent studies have suggested that tumor cells disseminate early on and develop the capacity to metastasize independently from the primary tumor. These circulating tumour cells (CTCs) represent the intermediate cells between primary tumours and metastases. The presence of CTCs in blood is an established prognostic marker of shorter progression-free and overall survival. It is currently impossible to distinguish CTCs from normal epithelial cells. They are also a rare population (approximately 1 out of 109 blood cells), making genomic profiling unachievable thus far. If the whole genome of CTCs can be screened for genomic alterations, two fundamental problems can be addressed (a) identifying a gene signature describing specific genomic alterations in early breast cancer that are associated with metastasis; and (b) utilizing this signature in the development of specific markers for CTCs in blood. Materials and Methods: We have successfully designed a protocol for the isolation of CTCs from blood for subsequent whole genome amplification (WGA) and microarray analysis. Blood samples from healthy donors were spiked with MCF7 tumor cells, and then enriched by automated immunomagnetic column separation. Enriched cell smears were stained for cytokeratin, using a glucose oxidase (GO) detection system. GO is absent from mammalian cells, which abolishes false positives seen with alkaline phosphatase and horse raddish peroxidase detection. Positively stained cells were isolated by single-cell laser capture microdissection, followed by WGA. Results: Genomic amplification was observed from as few as 2 MCF7 cells; with a sufficient yield of 1.5-3 μg of DNA. Microarray analysis was carried out on the high density Affymetrix Genome Wide SNP 6.0 array. Expected regions of amplification and deletion in the MCF7 cell line were identified in WGA samples. Copy number states were found to be strictly conserved across samples with single cell starting material (P&amp;lt;0.0001). We achieved up to 84% SNP call concordance between amplified single cell DNA and unamplified genomic DNA (P&amp;lt;0.0001). Discussion: We are isolating CTCs from the peripheral blood of 60 patients with early breast cancer. We have isolated CTCs and amplified DNA from 11 of 20 patients recruited so far. This preliminary study shows regions of DNA amplification that are unique to CTCs. We hypothesize that genes within these regions have the potential of being developed into CTC specific markers, and include genes associated with epithelial-mesenchymal transition, dormancy, cancer cell-stemness, migration, and invasion of the extracellular matrix. Identification of novel genomic alterations in CTCs associated with metastasis will pave the way for the development of a robust molecular or immunohistochemical prognostic test in patients with early breast cancer, to better identify those patients whose disease will progress to metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-09.

Persistence of BCR-ABL-Expressing LEUKEMIC STEM CELLS IN Chronic MYELOID LEUKEMIA (CML) PATIENTS IN COMPLETE REMISSION with Undetectable MOLECULAR Disease

AbstractAbstract 883Currently Imatinib Mesylate (IM) represent the first line therapy for chronic myeloid leukemia (CML). Recent data suggest that despite unprecedented rates of complete cytogenetic responses (CCR) and major molecular responses (MMR) obtained, leukemic stem cells (LSC) persist in the majority of patients (pts). LSC have been shown to be resistant to in vitro treatments with tyrosine kinase inhibitors (TKI). Consequently, discontinuation of IM in pts with undetectable molecular residual leukemia (UMRL) attested by RQ-PCR, leads to molecular relapses in the majority of the cases. Although the persistence of CD34+ CD38- leukemic stem cells has been demonstrated in pts with complete cytogenetic remission (CCR), the persistence of BCR-ABL+ leukemic stem cells in UMRL pts with has not been studied so far.For this purpose, we have performed an extensive analysis of bone-marrow-derived clonogenic and primitive hematopoietic stem cells in 6 pts with RQ-PCR constantly negative in their blood samples. Concerning the treatments; 5 out of 6 pts were off therapy, 3 pts (UPN1, 2, 3) had been treated with interferon-a only (IFN) for 6–13 years and their therapy was discontinued for 11, 16 and 8 years, respectively and 2 pts (UPN4 and 5) had been treated successively with IFN and IM and their IM therapy was discontinued for 2 years. One patient (UPN6) had been treated with IM followed by dasatinib and was on dasatinib at the time of the study. UPN7 was previously treated with first IFN then IM (which induced a stable UMRL) and then she switched to dasatinib because of side effect with IM. Bone marrow cells were collected and CD34+ cells purified using immunomagnetic columns. After performing a clonogenic assay, CD34+ cells were used in long-term culture initiating cell (LTC-IC) assays with weekly half medium changes. At week+5, clonogenic assays were performed and LTC-IC-derived clonogenic cells activity was calculated. For each patient 20 individual and 20 pools of 10 clonogenic cells and 20 individual and 20 pools of 10 LTC-IC derived CFU-C were plucked in order to obtain information in at least 220 CFU-C. After RNA extraction, BCR-ABL was quantified by RQ-PCR and in each positive CFU-C a nested PCR was performed to confirm the results. In one patient (UPN7) a NOD/SCID mouse assay was performed.All 3 pts treated with IFN alone, had BCR-ABL+ clonogenic cells varying from 0.5% (UPN1, 2) to 45 % (UPN3). All 3 had LTC-IC derived CFU-C positive for BCR-ABL (UPN1: 20%; UPN2 5%; UPN3 3%). In two pts previously treated with IFN and IM, clonogenic CFU-C BCR-ABL positivity was 10% and 5% whereas LTC-IC-derived CFU-C was 5% in UPN4) and undetected on UPN5. In UPN6 treated with IM then dasatinib, 5% of CFU-C was BCR-ABL+ whereas 100% of LTC-IC derived CFU-C was positive. The analysis of SCID-NOD assays performed in CD34+ cells from patient UPN7 is ongoing. Overall, these data show, for the first time to our knowledge, that in pts in IFN and IFN/IM-induced long-term remissions, there is persistent clonogenic BCR-ABL+ output maintained by BCR-ABL-expressing stem cells in the absence of relapse. In the only patient with successively treated with IM and dasatinib, 100 % of primitive hematopoietic stem cells are BCR-ABL+, despite PCR-negativity in peripheral blood, suggesting their possible quiescence in vivo and highlighting a theoretical risk of relapse. It remains to be determined if in pts with TKI-induced remissions, the analysis of stem cell compartments could be of use for clinical decisions to discontinue therapy. Disclosures:No relevant conflicts of interest to declare.

The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.

AbstractAbstract 3860 Objective.Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression. Materials and Methods.Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility. Results.Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers. Conclusions.Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers. Disclosures:No relevant conflicts of interest to declare.

Abstract 2120: Single cell whole genome amplification for high density SNP analysis of circulating tumor cells in early breast cancer

Abstract Despite progressive advances in the fields of radiation and chemotherapy, metastasis remains the leading cause of death in women with recurrent breast cancer. In metastasis, a small, select group of cells disseminate from the primary breast tumor, and circulate via the vascular system to distant organs, developing tumors at these new sites. Recent studies have suggested that tumor cells disseminate early on and develop the capacity to metastasize independently from the primary tumor. Furthermore, the enumeration of circulating tumour cells (CTCs) in blood is an established prognostic marker whereby, patients with more than 5 CTCs per 7.5mL of blood have shorter progression-free and overall survival. It is currently impossible to distinguish CTCs from normal epithelial cells. If the whole genome of CTCs can be screened for genomic alterations, two fundamental problems can be addressed (a) identifying a gene signature describing specific genomic alterations in early breast cancer that are associated with metastasis; and (b) utilizing this signature in the development of specific makers for CTCs in blood. We have designed a protocol for the isolation of tumor cells from blood for subsequent whole genome amplification (WGA) and microarray analysis. Blood samples from healthy donors were spiked with MCF7 tumor cells, and then enriched by automated immunomagnetic column separation. Enriched cell smears were stained for cytokeratin, using a glucose oxidase (GO) detection system. GO is absent from mammalian cells, which abolishes false positives seen with alkaline phosphatase and horse raddish peroxidase detection. Positively stained cells were isolated by single-cell laser capture microdissection, followed by WGA. Genomic amplification was observed from as few as 2 MCF7 cells; with a sufficient yield of 1.5-3 µg. Microarray analysis was carried out on the high density Affymetrix Genome Wide SNP 6.0 array. Expected regions of amplification and deletion in the MCF7 cell line were identified in WGA samples, as well as conserved across samples with single cell starting material (p&amp;lt;0.0001). Concordance of 75% was achieved from WGA of 3 single cells (p&amp;lt;0.0001). We propose to isolate CTCs from peripheral blood of 60 patients seen at the Locally Advanced Breast Cancer clinic at Princess Margaret Hospital. We have amplified genomic DNA from 6 out of 12 patients recruited so far, each having between 3-10 CTCs. Matched normal samples were obtained from the residual WBC pellet of each patient to allow for paired analysis. Optimization of WGA concordance will be followed by analysis of CTC DNA obtained from patients. Identification of novel genomic alterations in CTCs associated with metastasis will pave the way for the development of a robust molecular or immunohistochemical prognostic test in patients with early breast cancer, to better identify those patients whose disease will progress to metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2120.

In Vitro and In Vivo Study of Synergistic Effect of Imatinib and Simvastatin in Chronic Myelogenous Leukemia.

Abstract 2202Poster Board II-179 Background:Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods:Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results:Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion:We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures:No relevant conflicts of interest to declare.

An Inducible Caspase 9 Suicide Gene to Improve the Safety of Mesenchymal Stromal Cell Therapies.

Abstract 1444Poster Board I-467Given their immunomodulatory properties and potential for tissue differentiation, mesenchymal stromal cells (MSCs) are attractive vehicles for the treatment of graft-versus-host disease and autoimmune disorders and for regenerative cellular therapies. While MSCs have been infused in hundreds of patients to date with minimal reported side effects, follow-up is limited and little is known of their longer term potential for autonomous growth or unwanted activity or differentiation. Several in vitro and animal models have recently raised safety concerns, including reports of spontaneous osteosarcoma formation in culture, and ectopic ossification and calcification foci in the myocardium and lung. In light of those concerns, we sought to develop a system that could control the growth and survival of MSCs used therapeutically.We previously described a suicide gene system for T lymphocytes which was based on an inducible caspase-9 (iCasp9) protein that can be activated using a specific chemical inducer of dimerization (CID), which has been safely tested in a phase I study. Because caspase 9 should induce apoptosis even in differentiated and non-dividing cells, we tested this approach in MSC before and after induction of differentiation. MSCs isolated from healthy donors were transduced with a retroviral vector encoding iCasp9 together with a truncated CD19 (ΔCD19), to allow selection of transduced cells. After a single transduction, 47% ± 6% of the cells were iCasp9/CD19-positive, a percentage that was stable over more than two weeks in culture, suggesting no growth modulation of MSC by the construct. Transduced cells were readily selected by an immunomagnetic column to >97% purity. The phenotype of the iCasp9/CD19-positive cells was identical to that of untransduced cells, with >98% cells positive for CD73, CD90 and CD105, and negative for hematopoietic markers. Non-transduced MSCs had a spontaneous rate of apoptosis in culture of approximately 18% (±7%), and this was not increased following iCasp9-ΔCD19 transduction and selection (apoptotic rate 15% ± 6%, P = 0.47). Addition of CID to MSC cultures after transduction and selection with iCasp9-ΔCD19 resulted in the apoptotic death of 93% ± 1% of iCasp9-positive cells within 24 hrs (P < 0.0001 compared to control), while iCasp9-negative cells retained an apoptosis index similar to that of non-transduced controls (20% ± 7%, P = 0.99 and P = 0.69 vs. non-transduced controls with or without CID, respectively). Furthermore, iCasp9-positive MSCs injected subcutaneously in immunodeficient mice were selectively eliminated after administration of CID to the animals, as assessed by in vivo imaging, without systemic toxicity. Hence, iCasp9-positive MSCs can be selectively killed by exposure to this drug in vitro or in vivo. Moreover, addition of CID to cultures of MSCs differentiated to adipocytes, osteoblasts or chondroblasts also resulted in >90% apoptosis as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.In summary, we have demonstrated that MSCs can express an inducible caspase gene without affecting their phenotype, survival or capacity to differentiate, that the transduced cells can be selected with clinical grade procedures and maintain their basic physiology, and that both the MSC and their differentiated progeny can be selectively eliminated in vitro and in vivo by exposure to a small dimerizer molecule. This approach may provide an added margin of safety to the increasing clinical applications of MSCs and their progeny. DisclosuresNo relevant conflicts of interest to declare.