ALBERT

Description: “Fetal” erythrocytes are present in older children and certain adults with hematologic disorders. To determine if regenerating bone marrow produces such cells, we examined the blood of seven allogeneic bone marrow transplant recipients. Six patients were engrafted with donor cells, while on e patients recovered autologous bone marrow after rejection of a marrow transplant. All seven patients had fetal hemoglobin levels of up to 10% by 100 days after transplant. In three patients, the Ggamma to Agamma ratio in the fetal hemoglobin was “newborn”, while in one it was “adult”. Gamma chain synthesis in blood and bone marrow never exceeded 20% of total non-alpha globin synthesis. The fetal hemoglobin was heterogeneously distributed in the cells. High titer i antigen also appeared. All fetal characteristics declined by 200 days. Erythropoiesis during bone marrow recovery appears to be associated with an accelerated, albeit partial, recapitulation of ontogeny.

Description: Neutrophils play a vital role in the immune defense, which is evident by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. Both the GM-CSF–permitted survival of mature neutrophils and the generation of granulocytes from granulocyte-macrophage progenitors (GMPs) were markedly reduced in Stat5a/b mutants. GMPs showed impaired colony-formation ability with reduced number and size of colonies on GM-CSF stimulation. Moreover, continuous cell fate analyses by time-lapse microscopy and single cell tracking revealed that Stat5a/b-null GMPs showed both delayed cell-cycle progression and increased cell death. Finally, transcriptome analysis indicated that STAT5A/B directs GM-CSF signaling through the regulation of proliferation and survival genes.

Description: FVIItta/− mice are heterozygous for the FVII- null allele and a gene-targeted allele expressing very low levels of FVII. These mice produce very low levels of FVII (16 weeks) FVIItta/− males were selectively backcrossed with C57Bl/6J FVII+/− females to generate F1B2, F1B3 and then F1B4 FVIItta/− mice. Presumably, the survival of these FVIItta/− mice depends upon the retention of 129X1/svj derived chromosomal regions enabling the survival of low FVII mice. After 4 rounds of backcrossing, the F1B4 FVIItta/− mice retain ~3% of the 129X1/svj genome. DNA from 6 F1B3 FVIItta/− males and all their 81 F1B4 FVIItta/− offspring (total of 87 mice) have been genotyped with a Mouse MD Linkage panel (Illumina) containing 247 informative SNPs distinguishing between 129X1/svj and C57Bl/6J genomes. The initial scan identified 12 chromosomal regions enriched for 129X1/svj derived sequences. To identify putative hemostatic modifier genes, long-term surviving F1B4 low FVII mice have been selectively backcrossed and the DNA of their offspring analyzed with a high density SNP scan in the regions of interest.

Description: Neutrophils, one kind of granulocytes, are the most abundant type of white blood cells in human peripheral blood and form an integral part of the immune system. In addition, the majority of acute myelogeneous leukemia (AML) cells are from the granulocyte lineage. Granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) control migration, proliferation and survival of granulocytes. G-CSF and GM-CSF activate the transcription factors STAT5A/B (STAT5), which are essential for the development of T and B cells and the erythroid lineage. However, it is not clear to what extent G-CSF or GM-CSF signaling through STAT5 controls the differentiation, proliferation, survival in granulocyte lineage. STAT5 is not only essential for normal development and its constitutive activation has been linked to AML patients with Flt3 mutations. The objective of this study was to explore the contribution of STAT5 in G-CSF- and GM-CSF-induced granulopoiesis and to elucidate the underlying molecular mechanisms. Towards this goal, the Stat5a/b genes were deleted in mouse hematopoietic stem cells in vivo using Cre-loxP-mediated recombination (mutant mice). Injection of 5-FU resulted in a cytokine storm, which in controls, but not in mutant mice, led to a 10-fold elevation of neutrophils. Strikingly, the distribution of myeloid progenitor populations in bone marrow was not altered in STAT5-null animals in homeostasis. Colony assays were performed to address which cytokine controls granulopoiesis from these progenitors. While common multipotent progenitor cells (CMPs) and granulocyte macrophage progenitor cells (GMPs) from control mice formed large colonies in the presence of GM-CSF, mutant cells responded poorly. No difference between control and mutant colonies was observed in the presence of G-CSF. To investigate GM-CSF-mediated survival, apoptosis-assays were performed with peritoneal neutrophils. Greatly elevated apoptosis was observed with STAT5-null neutrophils. To further dissect the contribution of apoptosis and/or proliferation in the observed defects, long-term time-lapse imaging and single cell tracking was applied. Control and STAT5-null GMPs were cultured with GM-CSF and individual cells and all their progeny were continuously observed for 5 generations. Despite an equal number of initial GMPs responding to GM-CSF, the generation time of STAT5-null GMP-derived progeny was significantly prolonged in each generation and the number of cell death events increased dramatically from generation to generation. Therefore, GM-CSF-mediated STAT5 signaling is necessary to generate high numbers of granulocytic cells from GMPs by providing pro-survival and pro-proliferation signals. To identify GM-CSF-mediated and STAT5-dependent genetic cascades that control proliferation and survival of the granulocyte lineage, we performed gene expression profiling and ChIP-seq of control and STAT5-null CMPs, GMPs and neutrophils. STAT5 target genes specific to CMPs, GMPs and neutrophils were identified and their contribution to normal granulopoiesis is currently being investigated.

Description: “Fetal” erythrocytes are present in older children and certain adults with hematologic disorders. To determine if regenerating bone marrow produces such cells, we examined the blood of seven allogeneic bone marrow transplant recipients. Six patients were engrafted with donor cells, while on e patients recovered autologous bone marrow after rejection of a marrow transplant. All seven patients had fetal hemoglobin levels of up to 10% by 100 days after transplant. In three patients, the Ggamma to Agamma ratio in the fetal hemoglobin was “newborn”, while in one it was “adult”. Gamma chain synthesis in blood and bone marrow never exceeded 20% of total non-alpha globin synthesis. The fetal hemoglobin was heterogeneously distributed in the cells. High titer i antigen also appeared. All fetal characteristics declined by 200 days. Erythropoiesis during bone marrow recovery appears to be associated with an accelerated, albeit partial, recapitulation of ontogeny.

Description: Nonmyeloablative conditioning regimens followed by allogeneic hematopoietic cell transplantation (HSCT) rely on pre and post transplant immunosuppression to overcome host-versus-graft reactions and control graft-versus-host-disease (GVHD). Patients with less than 50% of donor T cells by day 14 after transplant have a higher risk of graft rejection and relapse than those with more than 50% donor chimerism (Blood2004; 104(8):2254). However, with increasing donor T-cell chimerism the risk of acute GVHD increases. Immunosuppression may inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells. Genetically modifying T cells with a drug resistance gene is one way to confer protection from the effects of the immunosuppressive agents, retain donor T cell function, and increase donor T cell chimerism by day 14. Mycophenolate mofetil (MMF) has been used in the post transplant setting to decrease the severity of GVHD and reduce the risk of host vs. graft reaction leading to graft failure. The active metabolite of MMF, mycophenolic acid (MPA), inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH). The type II isoform of IMPDH is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells which lack the salvage pathway for guanosine synthesis. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthetic activity ex vivo (BBA2002;1594:27, JBC.1997;272:961, BBA.1994;1217:156). We have generated six mutants of IMPDH II based on in vitro data and x-ray crystallography of the MPA binding site and transduced them into a human T cell line (Jurkat), a canine large granular lymphocyte cell line (CLGL-90) and a fibroblast cell line (NIH3T3) using oncoretroviral transduction. The mutants include: S276A (Amut), S276F (Fmut), T333I + S351Y (IYmut), L30F + Q277R (FRmut), Q277R + A462T (RTmut), and S276Y (Ymut). We generated bicistronic (IRES containing) retroviral vectors that expressed either HA-tagged wild type human IMPDH II (WT) or an HA-tagged IMPDH mutant and EGFP. We show that forced expression of WT will increase the MPA IC50 3 fold (IC50=3uM) compared to non-transduced cells. Forced expression of only the IY mutant in all cell lines tested was able to transfer resistance to MPA in excess of WT (IC50〉50uM). This is clinically relevant since MPA concentrations measured after transplant in patients on MMF are around 8uM (Blood.2005;106(13):4381). To show that resistance was related to the expression of the mutant enzyme we correlated GFP by FACS with HA-IMPDH II expression by western blot analysis, and correlated this with resistance to MPA in vitro. This was true regardless of selection in MPA or transduction and cell sorting for 〉90% GFP positive cells. Analysis of IMPDH activity in lysates of transduced cells is currently underway. This is the first report where forced expression only of the IY mutant provides a clear enhanced resistance to MPA in vitro compared to cells over expressing wild type IMPDH II. Currently, we are generating bicistronic maloney based oncoretroviral vectors which carry a suicide gene (CD34-TK) and the IMPDH-HA IY mutant which will allow us to pharmacologically modulate the level of donor T cells after allogeneic stem cell transplantation.

Description: Abstract 3378 Poster Board III-266 Introduction: Haploidentical-cord blood transplantation is a promising approach for patients (pts) who lack HLA donors and may improve rates of early engraftment while allowing long term cord blood reconstitution with low rates of GVHD. We enrolled 29 pts (17 AML/MDS, 4 ALL, 3 CML, 4 NHL/HL, 1 severe aplastic anemia) lacking HLA identical donors. The median age was 40 years (range, 4-67), and median weight was 75 kg (range, 14-125). Twenty-two (76%) pts had active disease at time of transplant; 6 had prior autologous transplants. 14 pts were Caucasian; 15 were other race or ethnicity. The haploidentical donor was the mother in 4; father in 3; child in 10; sibling in 10; and half-sibling in 2 cases. The median haploidentical CD34+ dose was 2.51 × 106/kg (range, 1.25-10.95); CD3+ cells were 1.0 × 104/kg (range, 0.3-3.7). Single unrelated CB units were matched by low resolution at HLA-A and B and high-resolution at DRB1, and matched 6/6 in 2 pts; 5/6 in 18 pts; 4/6 in 9 pts. Median cord total nucleated cells equaled 1.93 × 107/kg (range, 1.07-9.36); CD34+ cells were 0.08 × 106/kg (range, 0.03-0.75). The conditioning regimen for 18 pts was fludarabine (Flu) (30 mg/m2 on d-7 through -3), melphalan (Mel) (70 mg/m2 on d -3 and -2), and Thymoglobulin (rATG) (1.5 mg/kg on d-7, -5, -3, -1). Eleven pts received Flu, thiotepa (5 mg/kg on d -7 and -6), total-body irradiation (TBI) (12 Gy lateral opposed fields in 2 Gy fractions BID on d-3 through -1), and rATG. GVHD prophylaxis consisted of tacrolimus (Tac) + methylprednisolone or Tac + mycophenolate. Engraftment: Two pts died early (sepsis, CVA). Three other pts failed to engraft with either haploidentical or CB and died of infection on d36, 43, and 63. One of these had anti-donor HLA antibodies. 24 pts engrafted with a median time to ANC 〉500/mL of 10 days (range, 9-31) and median time to sustained platelets 〉20,000/mL of 20 days (range, 15-63). In the majority of pts, early haploidentical engraftment was replaced by durable engraftment of CB by 100 days. However, 3 pts had persistent hematopoiesis associated with only the haploidentical donor, while a fourth pt engrafted with only CB on day 31. Late graft failure and death from sepsis occurred in one of the patients with haploidentical engraftment. In unfractionated peripheral blood or bone marrow cells, median haploidentical chimerism was 95% (range, 0-100) on d14; 76% (range, 0-95) on d30; 6% (range, 0-87) on d100. Median unfractionated cord chimerism was

Description: Abstract 1039 Poster Board I-61 Background: The inhibitor of apoptosis protein (IAP) survivin is central in integrating proliferative and cell cycle regulatory networks. In leukemic cells, survivin mediates survival as well as resistance to chemotherapeutics and Flt-3 inhibitors. Experimentally targeting survivin has shown anti-leukemic activity and is a postulated therapeutic approach. Herein we report a clinical trial with the novel survivin and cdc2 (CDK1) inhibitor Terameprocol (EM-1421) in patients (pts) with advanced hematological malignancies. As little is known about in-vivo up- and downstream regulatory pathways of survivin and it's inhibition, specimens from pts were collected for correlative pharmacodynamic (PD) marker analysis. Methods: Open-label, single agent, phase I dose escalation study of Terameprocol (T) in pts with advanced, relapsed or refractory hematological malignancies (AML, ALL, MDS, advanced CLL or CML). Pts age 〉 18 years with adequate organ function and performance status (ECOG) ≤ 1were treated with 1000, 1500 and 2200 mg of intravenous Terameprocol 3x/week (wk) for 2 of 3 wks in cohorts of 3 pts to establish the safety, maximum tolerated dose (MTD) and to assess pharmacokinetics at the studied dose schedule (primary objectives). Secondary objectives were to select the recommended phase 2 dose (RP2D) and to assess anti-leukemic activity and PD marker regulation (baseline, cycle 1 day 5 and 12, end of study). Results: Between 8/2007 and 3/2009, sixteen pts (4 female, 12 male) with a median age of 68.5 years (range 42-78) and median of 2 prior regimens (range 0-5) were enrolled. Most pts had AML (n=13), 7 pts primary and 6 pts secondary or treatment related AML; one pt each had CML-BP, T-ALL and MDS. Ten pts had unfavorable or complex cytogenetics including 6 pts with 5q/7q and 2 pts with 11q23 aberrations. Four, 5 and 6 pts were treated at the 1000, 1500 and 2200 mg dose cohorts respectively. One pt did not start treatment on study. 15 pts received ≥ 1 dose/cycle, 6 of whom (38%) received ≥ 2 cycles of T (range 1-5). Common possible or probable treatment related adverse events (AE) were grade 1 or 2 headache (n=3, 20%), transaminitis (grade 2 n=2, grade 3 n=2) and pruritus (n=2). Treatment related serious AE's (SAE) was a grade 4 pneumonia in 1 pt. Non-drug related SAE's ≥ grade 3 or 4 included sepsis/febrile neutropenia (n=3), pneumonia (n=2), dyspnea (n=2), cerebral hemorrhage (n=1), confusion/mental status change (n=1), cardiac arrest (n=1) and AML progression (n=3) leading to death in 2 pts. No AE/SAE was felt to constitute a dose limiting toxicity (DLT) per protocol definition. However, due to grade 3 transaminitis observed in 2 pts together with concerns of compromised respiratory status of pts treated at the 2200 mg cohort, the investigators determined the maximum tolerated dose (MTD) to be 1500 mg 3x/week for 2 of 3 weeks, which is also the recommended RP2D. One heavily pretreated pt (3 prior regimens) with CML-BP myeloid, achieved a partial remission (1500 mg) and transfusion independence for 5 cycles prior to disease progression. Hematological improvement (HI-E, HI-P) was seen in 1 pt (1000 mg), and 5 pts had stable disease. Surprisingly, Cmax of T at 1500 mg was higher than at 2200 or 1000 mg. Overall concentrations of T were in the same range as measured in previous studies of T in solid tumors (daily x5), indicating adequate drug exposure at the schedule studied. PD samples at indicated time points were collected and are currently being analyzed to assess the effects of T on survivin, cdc2/CDK1 and survivin associated regulatory genes. Conclusion: The novel small molecule surivin inhibitor Terameprocol can be safely administered to pts with advanced leukemias. Sufficient drug exposure was seen in pts and the MTD and RP2D were established for future studies. Clinical activity was observed in a pt with myeloid CML-BP and potentially in pts with AML. Interestingly, previous work showed an association between progression to advanced stages of CML and survivin expression. Data on correlative PD marker experiments will be presented. Disclosures: Tibes: Erimos Pharmaceuticals : Research Funding. McDonagh:Erimos: Research Funding. Frazer:Erimos Pharmaceuticals: Employment. Mohrland:Erimos Pharmaceuticals: Employment. Von Hoff:Erimos Pharmaceuticals: Consultancy.

Description: Abstract 197 Supported by an unrestricted grant from Genzyme Corporation. Fludarabine (Flu) melphalan-alemtuzumab is a well tolerated, reduced intensity conditioning regimen for HCT. Clofarabine (Clo), a second generation nucleoside analog with excellent activity in acute leukemia, might enhance disease control over Flu. We report outcomes of a completed phase I and ongoing phase II study of CMA conditioning for allogeneic peripheral blood HCT. Tacrolimus was administered as GVHD prophylaxis. For the phase I cohort, one pt was enrolled per dose level, until the first DLT or until 2 had grade 3 toxicity. Dose level 1 consisted of: clo 10 mg/m2/day on d −7 to −3 and melphalan 100 mg/m2 on day −2. Clo was increased by 10 mg/m2/day per cohort until 40 mg/m2/day. Then, melphalan was increased by 20 mg/m2 until 140 mg/m2. Alemtuzumab was given at a fixed dose of 20 mg/day on d −7 to −3. Twelve pts were accrued in the phase I portion of whom three remain in remission at 26, 22 and 21 months. Forty pts, median age 53 (24–69), have been accrued in the phase II study of whom 16 had related and 24 unrelated donors. 20 had AML/MDS (6 refractory, 4 CR2, 9 CR1, 1 untreated MDS), 16 NHL (6 refractory, 9 chemosensitive relapse, 1 CR1) 2 CLL, 2 MPD. ASBMT risk score was high in 14, intermediate in 14, and low in 12. Performance score was 0 in 19, 1 in 17, 2 in 2, and not documented in 2 patients. The phase II dose was initiated at Clo 40 mg/m2/day x 5 days and melphalan 140 mg/m2. Twenty-four pts received this dose. Grade 3 renal toxicity occurred between day −7 and day +7 in 4 of 24 (17%) pts receiving this dose. The phase II dose was then reduced to Clo 30 mg/m2/day x 5 days and melphalan 140 mg/m2, and used to treat 16 pts. One pt with preexisting cardiomyopathy and refractory AML died during conditioning from cardiovascular failure. No grade 3 renal toxicity has been observed in this cohort and 3 pts had reversible grade 2 renal failure. Other toxicities included: gr 2–3 reversible ALT elevation between day −2 and day +5 in 8 pts; gr 2 reversible bilirubin elevation in 1 pt. No grade 3–4 hand foot syndrome or VOD occurred in this cohort. All evaluable pts engrafted. Twenty of 24 pts had full donor CD3 chimerism on day 30 and 2 had mixed donor chimerism. 11 pts had gr II aGVHD, and 3 had gr IIII/IV aGVHD. 7 have cGVHD.With a median follow-up of 313 days (19–607), 24 of 40 pts (60%) in the phase II portion of the study remain in remission. Eight have relapsed, 4 of whom have died. Eight others have died of treatment-related causes (7 after Clo 40 and 1 after Clo 30). Estimated one year survival is 72% (95%CI, 56–88) and PFS is 63% (45–81%). Neither dose of Clo (40 vs 30), donor type (MUD vs related), age (〈 50 vs 〉50) affected outcomes. One year PFS was 56% (28–84) for NHL and 68% (46–90) for AML/MDS (P=NS). One year PFS was 43% (15–71%) for ASBMT high risk pts vs 70% (50–90%) for ASBMT low/intermediate risk pts (P=0.01; Figure 1). Conclusions: Clofarabine - melphalan - alemtuzumab conditioning induces durable remissions in a substantial fraction of patients with advanced hematologic malignancies. Clo 30/Mel 140 has an excellent safety profile. Disclosures: Off Label Use: clofarabine for transplant conditioning. Kline:Genzyme corporation: Membership on an entity's Board of Directors or advisory committees. Odenike:Genzyme corporation: Membership on an entity's Board of Directors or advisory committees. Stock:Genzyme: Research Funding.

Description: Among 2333 consecutive patients with myelofibrosis with myeloid metaplasia (MMM) seen at our institution, 91 fulfilled the World Health Organization (WHO) criteria for leukemic transformation (LT). All episodes of LT were myeloid in origin (acute myeloid leukemia [AML]) with all French-American-British (FAB) subtypes represented except M3; the most frequent subtypes were M7 (25.4%), M0 (22.4%), and M2 (17.9%). Cytogenetic studies during LT were available in 56 patients and revealed a clonal abnormality in 51 (91%): 30 patients had complex karyotype, 2 had core-binding factor gene lesions, and 18 had abnormalities of chromosome 5 or 7. Karyotypic evolution was documented in the majority of the patients in whom serial analysis was possible. In general, LT was fatal in 98% of the cases after a median of 2.6 months (range, 0-24.2 months). Twenty-four patients received AML-like induction chemotherapy that resulted in no complete remission: 41% reverted into chronic-phase disease and the incidence of treatment-related mortality was 33%. The remaining 67 patients received either supportive care alone (48 patients) or low-intensity chemotherapy (19 patients). Overall, survival was similarly poor in all 3 treatment categories. The outcome of LT in MMM with current therapies is dismal and either supportive care alone or appropriate clinical trials should be considered.