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We have previously reported that expression of the erythropoietin (Epo) gene in mouse embryonal cells was not induced by hypoxia, although hypoxia induced other hypoxia-inducible genes. This study identifies retinoic acid (RA) as an inducer for Epo production in the embryonal carcinoma cell lines P19 and F9. RA induced Epo production through the transcriptional activation of the Epo gene in an oxygen-independent manner. With the use of reporter assays in P19 cells, it is shown that a direct repeat of the nuclear hormone receptor-binding motif separated by a 2-bp spacer (DR-2) in the hypoxia-response enhancer was responsible for the transcriptional activation by RA. Electrophoretic mobility shift assays show that nuclear extracts from P19 cells contained RA receptor complexes that bound to DR-2. In human hepatoma Hep3B cells, an orphan receptor, hepatocyte nuclear factor-4, strongly augmented hypoxic induction of the Epo gene in cooperation with hypoxia-inducible factor-1 (HIF-1) by binding to DR-2, whereas in P19 cells, the interaction of RA receptors with DR-2 was sufficient for RA-induced transcriptional activation of the Epo gene without the requirement of the HIF-1 site. These results suggest that DR-2 regulates expression of the Epo gene by acting as the binding site for different transcription factors in different types of cells.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM- CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13- acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.

Abstract 4342 (Purpose) We retrospectively analyzed the data regarding patients who received fractionated administration (FR) of gemtuzumab ozogamicin (GO) and standard administration (ST) of GO for the treatment of their refractory and relapsed acute myeloid leukemia in order to review the efficacy and safety of GO in each type of administration. (Method) The patients with relapsed and refractory acute myeloid leukemia (excluding patients with acute promyelocytic leukemia) who were treated with the ST of GO received 2 doses of monotherapy with 9 mg/m2 GO as a 2-hour intravenous (iv) infusion with a 14–28-day interval. The fractionated GO group received 2 doses of GO monotherapy administered as a 2-hour iv infusion of 3 mg/(m2·day) on days 1, 3, and 5 with a 14–28-day interval. (Result) Eleven patients received ST and 9 received FR. The median age of all patients was 66.1 (55–77) years. Overall response rates (CR+CRp) in the ST and FR groups were 27% and 33%, respectively. The adverse events (grade 3–4) in the ST and FR groups were neutropenia (100% vs. 100%), thrombocytopenia (90.9% vs. 88.9%), and hypertransaminasis (18.2% vs. 0%). Grade 3–4 hypertransaminasis was seen only in the ST group. Infusion reactions occurred in 5 patients: 3 in the ST group and 2 in the FR group. The median CD33 positivity of blast cells of patients who received CR or CRp was 90.9% (78.2–99.5%). (Discussion) The response rates of the ST and FR groups were similar. However, the incidence of grade 3–4 hypertransaminasis tended to be higher in the ST group. These data suggest that FR of GO is less toxic than ST. Thus, FR of GO is safer than ST even for Japanese patients with acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Background: Allogeneic stem-cell transplantation (allo-SCT) from human leukocyte antigen identical related donor (RD) is taken priority over unrelated donor (URD), however, less than 30% of patients do not have a suitable RD. Transplantation from an URD should be considered for those patients. In order to investigate the role of URD, we retrospectively analyzed efficacy and safety of allo-SCT from URD and RD in patients with myeloid malignancies. Patients and methods: One hundred six patients with myeloid malignancies (acute myeloid leukemia 48, chronic myeloid leukemia 38, and myelodysplastic syndrome 20) who received myeloablative regimens were analyzed. The stem cell source were bone marrow from URD (n=43) and peripheral blood stem cells or bone marrow from RD (n=63). Graft-versus-host disease (GVHD) prophylaxis consisted of taclorimus and short-term methotrexate (sMTX) for URD, or cyclosporine and sMTX for RD. Results: Median follow-up was 6.3 (0.3–17.2) years, and median age of patients was 37 (15–55) years old. Hematological studies demonstrated donor engraftment in all patients. Incidence of grades II to IV of acute GVHD was 32% in URD and 18% in RD, and extended chronic GVHD was 31% in URD and 28% in RD. Probability of 8-year actuarial survival was 62% in URD and 54% in RD (p=0.4225), and 8-year disease-free survival was 64% in URD and 47% in RD (p=0.1412). There were no apparent differences in transplant-related mortality (TRM) (14% in URD and 24% in RD), relapse (14% in URD and 32% in RD) between both groups. Conclusion: These results suggested that allo-SCT from URD has comparable safety and effectiveness for adult patients with myeloid malignancies.

Purpose: High-intensity conditioning regimens before allogeneic stem cell transplantation (all-SCT) could reduce relapse but may increase nonrelapse mortality. We have investigated the efficacy and feasibility of an intensive preparative regimen of busulfan (BU), cyclophosphamide (CY), and total body irradiation (TBI) for patients with myeloid malignancies. Patients and methods: From November 1990 to February 2005, 80 patients (acute myeloid leukemia (AML) 33, chronic myeloid leukemia (CML) 29, myelodysplastic syndrome (MDS) 18) treated with BU (8 mg/kg), CY (120 mg/kg), and TBI (10 Gy) followed by related (n=46) or unrelated (n=34) allo-SCT. Thirty-eight consecutive patients with AML in 1st remission, CML in chronic phase, and MDS (refractory anemia) defined as a standard risk group, and 42 patients with advanced AML, CML, and MDS (more than 5% marrow blasts) defined as a poor risk group. RESULTS: With a median follow-up was 6.9 years, the 7-year actuarial overall survival (OS) rate was 58% (standard risk 70% vs poor risk 47%, p=0.0076), and the probability of 7-year relapse rate was 18% (standard risk 17% vs poor risk 18%, p=0.4777). Nonrelapse mortality was 29% (standard risk 26%, poor risk 31%). Multivariate analysis indicated that age older than 40 years (p=0.018) and received allo-SCT before 1998 (p=0.006) had independent predictive value for nonrelapse mortality. CONCLUSION: The BU- CY- TBI regimen resulted in decreased relapse, especially in poor risk patients. The results of this study suggested that the BU- CY- TBI is an effective and feasible preparative regimen for allo-SCT in patients with myeloid malignancies.

Purpose: Acute lymphoblastic leukemia (ALL) remains a difficult disease to treat in adults and less than 40% of ALL patients could be alive at 5 years post-diagnosis. We investigated whether intensive conditioning regimens improve outcome of patients with ALL. Methods: Retrospectively, we analyzed 24 patients (median age; 29 years) with ALL who underwent allogeneic transplantation in our institute between February 1991 and October 2003. The median follow-up time was 7.7 years. Sixteen patients were in first complete remission (CR1) and five patients were in second CR (CR2) and three patients did not attain CR (non-CR) at the time of transplantation. Five patients with Philadelphia (Ph) chromosome-positive ALL were included. Preparative regimens were cyclophosphamide (CY), total body irradiation (TBI) and etoposide based regimen (n=23) or CY, TBI, and cytosine arabinoside (n=1) followed by marrow (n=21) or peripheral blood (n=3) transplant from unrelated (n=7) or related donors (n=17). Results: The 8-year overall survival (OS) rate and the 8-year disease-free survival rate for all cases were 48% and 39%, respectively. The 8-year OS of Ph negative and positive patients were 56% and 20%. The 8-year OS of CR1, CR2 and non-CR patients were 50%, 60% and 0%, respectively. Overall probability of the 8-year relapse rate was 47%. Treatment related mortality was 25%. Conclusion: These results suggest that CY/TBI based intensive regimens are feasible for ALL patients and may be effective for Ph negative ALL patients in not only CR1 but also CR2.

[Background] In JALSG Ph + ALL protocol(IDEAMOP) study, combination therapy with conventional intensive chemotherapy with imatinib (600mg/day) provided high quality of hematologic and cytogenetic (94%) and molecular (77%) complete remission (CR) for the patients with newly diagnosed Ph + ALL(Blood2004;104:748a #2736). However, a few of patients attained molecular CR relapsed subsequently, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) should be considered for younger patients as possible as they can. In order to investigate a role of allo-HSCT post IDEAMOP therapy, three patients treated in our institute were analyzed efficacy and toxicity of allo-HSCT. [Patients and Methods] Those three newly diagnosed Ph + ALL patients (M/F:2/1 Age:30,31,38 y.o.) were treated with IDEAMOP between Jan 28,2003 and Feb 13,2004. All patients attained CR after single course of induction therapy and median time of CR was 21 (19–27) days. During consolidation therapies, two of them found HLA-matched unrelated donors and one patient chose to receive cord blood transplantation. As conditioning regimen for allo-HSCT, all of them received total body irradiation, high-dose cyclophosphamide and high-dose etoposide before allo-SCT. [Results] Allo-HSCT was undergoing during their first CR(CR1). Regimen related toxicity and complications of the transplantation were well tolerated. The reconstitutions of bone marrow function were similar to other patients with Ph-negative ALL treated by allo-HSCT. Although two patients presented with either limited or extensive chronic GvHD, they were clinically controlled with or without steroid. Minimal residual disease (MRD), measured by RQ-PCR assay, was detected in two of three patients before allo-HSCT. However, MRD was not detected in any of the patients after allo-HSCT. All of three patients remain in CR at a median follow-up time of 25 (18–31) months. Allo-HSCT post JALSG-Ph+ALL (IDEAMOP)protocol Patient Status at HSCT MRD pre HSCT Cell Source MRD postHSCT cGvHD Outcome OS(M) 30Y M CR1 yes Cord Blood no limited alive 31+ 31Y M CR1 no MUD no extensive alive 25+ 38Y F CR1 yes MUD no extensive alive 18+ [Conclusion] Although the number of patients are small, imatinib (600mg/day) with intensive chemotherapy as remission induction for newly diagnosed Ph + ALL may have benefit for rapid control of disease, and may provide better outcome of allo-HSCT without additional severe toxicities.

Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Furthermore, for some chimeric transcription factors generated in leukemia-associated chromosomal translocations including PML-RARα and AML1-ETO, it has also been proposed that aberrant recruitment of corepressor complexes caused by oligomerization is critical for the development of leukemia. Among leukemia-associated transcription factors is Evi-1, which is frequently activated in cases with myeloid malignancies. Evi-1a and Evi-1c, with the latter also called MDS1-Evi-1, are two alternative forms derived from the Evi-1 gene. Evi-1a is a Zn finger transcription factor which recruits CtBP as a transcriptional corepressor and possesses the ability to repress TGF-β signaling. Amino-terminal to the Evi-1a, Evi-1c harbors an additional sequence similar to the PR domain, which was originally identified in PRDI-BF1 and RIZ1. In contrast to frequent upregulation of Evi-1a in myeloid malignancies, expression of Evi-1c widely varies depending on cases. Thus, although several lines of evidence suggest that Evi-1a is involved in leukemic transformation of hematopoietic cells, a role for Evi-1c in leukemogenesis has remained elusive. To elucidate the mechanism that underlies leukemogenesis by Evi-1, we investigated the ability of Evi-1 proteins to form oligomeric complexes. We found that Evi-1a forms a homo-oligomer in mammalian cells, whereas Evi-1c exclusively exists as a monomer. Remarkably, Evi-1c has lost the ability to interact with CtBP and failed to efficiently repress TGF-β signaling. We previously reported that AML1-Evi-1, a chimeric transcription factor generated in t(3;21) leukemia, interacts with CtBP as Evi-1a does. In the current study, we also found that AML1-Evi-1 forms a homo-oligomer, suggesting a close relationship between oligomer formation and CtBP binding. Taken together, these data indicate that homo-oligomerization may contribute to the oncogenic potential of the Evi-1 proteins by regulating the interaction with CtBP. These results also identify a novel function of PR domain to dictate oligomerization of transcription factors.

[Purpose] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the powerful therapeutic tools for patients with adult lymphoblastic leukemia (ALL) even though transplant-related toxicity and the recurrence of leukemia are still problematic. In order to improve outcome of patients with adult ALL who received allo-HSCT, we investigated the clinical efficacy and safety of etoposide containing conditioning regimen for allo-HSCT. [Patients and Methods] Adult patients (including patients with 17, 18 and 19 years of age) with ALL were treated between February 1991 and October 2003 in our institute. When patients attained complete remission (CR) by induction therapies using JALSG ALL protocols or others, they received allo-HSCT [bone marrow (BMT), peripheral blood stem cell (PBSCT), and cord blood transplantation (CBT)]. Conditioning regimen was consisted with etoposide (60 mg/kg), cyclophosphamide (120 mg/kg), and fractionated total body irradiation (10 Gy) (CY/ETP/TBI). As clinically indicated, cytosine arabinoside (CY/ETP/TBI/AraC) or nimustine hydrochloride (CY/ETP/TBI/ACNU) was added to CY/ETP/TBI. [Results] In the 23 patients [male/female: 15/8, median age was 30 years (range 17–50 years)], 19 patients received BMT (13 from related and 6 from un-related donor), 3 received PBSCT from related donor, and one was performed CBT. As the conditioning regimen, 16 patients received CY/ETP/TBI, 4 received CY/ETP/TBI/AraC, and 3 received CY/ETP/TBI/ACNU. Six out of 23 patients had Philadelphia chromosome positive ALL. Twenty-two patients received HLA serologically identical donor. Remission status at transplant included 16 patients in 1st CR, 5 in 2nd CR, and other 2 patients in non-CR. Grade II toxicity according to Bearman scale was observed in 52% (stomatitis), 22% (gastrointestinal), 9% (bladder), 4% (hepatic), and 4% (cardiac). Critical regimen-related toxicities and fatal veno-occlusive disease were not observed in any patients. The disease-free (DFS) and over-all survival (OS) rate of all patients was 51.1 % and 55.8 %. [Discussion/Conclusion] These results indicated that an intensified etoposide containing preparative regimen is a feasible regimen and it has good efficacy for transplant recipients with ALL.