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We conducted a prospective randomized study to assess the optimal postremission therapy for adult acute myeloid leukemia in patients younger than 65 years in the first complete remission. A total of 781 patients in complete remission were randomly assigned to receive consolidation chemotherapy of either 3 courses of high-dose cytarabine (HiDAC, 2 g/m2 twice daily for 5 days) alone or 4 courses of conventional standard-dose multiagent chemotherapy (CT) established in the previous JALSG AML97 study. Five-year disease-free survival was 43% for the HiDAC group and 39% for the multiagent CT group (P = .724), and 5-year overall survival was 58% and 56%, respectively (P = .954). Among the favorable cytogenetic risk group (n = 218), 5-year disease-free survival was 57% for HiDAC and 39% for multiagent CT (P = .050), and 5-year overall survival was 75% and 66%, respectively (P = .174). In the HiDAC group, the nadir of leukocyte counts was lower, and the duration of leukocyte less than 1.0 × 109/L longer, and the frequency of documented infections higher. The present study demonstrated that the multiagent CT regimen is as effective as our HiDAC regimen for consolidation. Our HiDAC regimen resulted in a beneficial effect on disease-free survival only in the favorable cytogenetic leukemia group. This trial was registered at www.umin.ac.jp/ctr/ as #C000000157.

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for acute myeloid leukemia (AML), but non-relapse mortality (NRM) remains obstacles to this treatments. Therefore, precise comorbidity evaluation before transplantation is crucial for better outcomes. One of the most frequently used tools for assessing the risk profile of transplant recipients is the hematopoietic cell transplantation comorbidity index (HCT-CI), which only incorporates information of the antibiotics use on day 0 as an infection-related factor, however, some important components, such as the number and severity of infectious episodes, are not included. Methods From December 2001 to December 2005, 1,057 newly diagnosed adult AML patients were registered to JALSG AML201 (Blood 2011; 117: 2358-65, 2366-72). Of them, the 121 evaluable patients who underwent allo-HSCT in the first hematological complete remission were analyzed. We have checked the above mentioned components, in addition to the interval from the last chemotherapy to transplantation, in these 121 patients and analyzed whether these factors might affect the subsequent transplant outcome. Results The median age was 37 years (range, 15-61). Regarding cytogenetic risk, 7 (6%), 92 (76%), and 15 (12%) patients were categorized as favorable, intermediate and high risk, respectively. The median follow-up period after transplantation was 939 days (range 19-2204). At 2 years after allo-HSCT, overall survival, leukemia free survival, cumulative incidence of relapse (CIR), and NRM for all patients were 73.2%, 65.4%, 19.4%, and 15.8%, respectively. A total of 106 (88%) patients developed at least one infectious episode as 102 (84%) patients faced to febrile neutropenia (FN), 45 (37%) patients experienced severe infection (≥ grade 3), and 27 (22%) patients eventually developed sepsis. Regarding the episode of FN, 52 (43%) patients experienced more than 2 times during chemotherapy. The median cycles of chemotherapy before transplantation was 4 (range, 1-6) and the median interval from the first day of last chemotherapy to transplantation was 77 days. The ROC curve indicated mild correlations between NRM and the interval with an area under the curve of 52% and the cutoff value at 113 days. The number of infectious episodes or severity of infection did not carry a significant impact on NRM at 2 years as 19.2% in FN with ≥3 episodes vs. 13.2% in FN with 0-2 episodes; p=0.18, 18.0% in patients with severe infection vs. 14.5% in patients without severe infection; p=0.49. However, in view of bone marrow transplant (BMT) recipients (n=76), multiple FN episodes was significantly associated with higher NRM (23.5% vs. 7.2% at 2 years, p=0.029) (Fig 1A). While, the relative shorter interval ( Conclusion Combining the number of infectious episodes and interval of chemotherapy to transplantation may predict a transplant outcome in AML patients. Providing an adequate interval from the last chemotherapy to transplantation may reduce NRM especially in BMT recipients who developed multiple FN episodes during induction or consolidation chemotherapy. Figure 1. Figure 1. Disclosures Kiyoi: Astellas Pharma Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Celgene Corporation: Research Funding; FUJIFILM Corporation: Research Funding; Phizer Japan Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Naoe:Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

It is generally recognized that postmitotic neutrophils give rise to polymorphonuclear neutrophils alone. We obtained evidence for a lineage switch of human postmitotic neutrophils into macrophages in culture. When the CD15+CD14- cell population, which predominantly consists of band neutrophils, was cultured with granulocyte macrophage–colony-stimulating factor, tumor necrosis factor-α, interferon-γ, and interleukin-4, and subsequently with macrophage colony-stimulating factor alone, the resultant cells had morphologic, cytochemical, and phenotypic features of macrophages. In contrast to the starting population, they were negative for myeloperoxidase, specific esterase, and lactoferrin, and they up-regulated nonspecific esterase activity and the expression of macrophage colony-stimulating factor receptor, mannose receptor, and HLA-DR. CD15+CD14- cells proceeded to macrophages through the CD15-CD14- cell population. Microarray analysis of gene expression also disclosed the lineage conversion from neutrophils to macrophages. Macrophages derived from CD15+CD14- neutrophils had phagocytic function. Data obtained using 3 different techniques, including Ki-67 staining, bromodeoxyuridine incorporation, and cytoplasmic dye labeling, together with the yield of cells, indicated that the generation of macrophages from CD15+CD14- neutrophils did not result from a contamination of progenitors for macrophages. Our data show that in response to cytokines, postmitotic neutrophils can become macrophages. This may represent another differentiation pathway toward macrophages in human postnatal hematopoiesis. (Blood. 2004;103:2973-2980)

Abstract 1963 Purpose: Management of febrile neutropenia in patients receiving allogeneic hematopoietic stem cell transplantation (HCT) is crucial for successful treatment, but precise pathogen identification is difficult because the sensitivity of blood culture remains low, making the antimicrobial therapy empiric. This study aimed to detect and identify bacterial and fungal pathogens from peripheral blood samples during early phase of HCT, using highly sensitive PCR method which is able to detect and identify a broad range of bacteria and fungi (J Clin Microbiol. 48(6):2030-6, 2010) and to assess the clinical usefulness of PCR analysis in managing febrile neutropenia after HCT. Patients and Methods: Ten consecutive patients who underwent HCT in our institute between June 2007 and December 2009 were enrolled. Eight patients received TBI-based conventional and two received reduced-intensity preparative regimens. All patients received antibacterial prophylaxis with fluoroquinolone and antifungal prophylaxis by mold-active azole with temporary use of micafungin. We prospectively performed PCR analysis and blood culture of blood samples at least once a week for the first 30 days of HCT. For species identification, positive PCR products were sequenced. Results: In a total of 56 analyses, bacteria were detected in 23 cases with PCR but in only one case with blood culture. In the febrile patients, 18 cases out of 37 PCR analyses were positive for bacteria, while, in afebrile cases, 5 cases out of 19 PCR analyses were positive for bacteria. In 17 pathogen identified cases, 14 were gram positive cocci and 3 were gram negative rods. Most of the identified pathogens were oral and intestinal bacteria reflecting grade 3 severe mucositis. Fever was manageable in most cases with empiric use of antibiotics, but several series of antibiotic change was required in 2 cases, in which bacteria susceptible to specific antibiotics such as Stenotrophomonas maltophilia were detected. In several cases suffering from severe diarrhea, pathogen could not be identified because PCR products formed multiple peaks. Fungi were detected in only 4 cases, of which 2 cases were provable Aspergillus infection and another 2 cases possessed no other evidence of fungal infection. Conclusion: Bacterial pathogens mostly associated with oral and intestinal mucositis were detected with PCR in approximately half cases of febrile neutropenia, but fever was manageable in most cases with empiric antibiotic therapy by experienced physicians. PCR analysis was considered to be useful in febrile neutropenia due to bacteria susceptible to specific antibiotics such as Stenotrophomonas maltophilia. More rapid and simplified approach which targets clinically important pathogens such as Stenotrophomonas maltophilia that requires specific use of antibiotics may be useful and reasonable to provide support for the choice of antibiotics in clinical practice. Disclosures: No relevant conflicts of interest to declare.

Abstract 3090 Poster Board III-27 The outcome of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has dramatically improved since the start of treatment with imatinib. The Japan Adult Leukemia Study Group (JALSG) has reported a high complete remission (CR) rate for Ph+ALL treated with imatinib-combined chemotherapy (Yanada et al, J Clin Oncol 2006). Here we report a follow-up analysis of the results of the JALSG imatinib-combined chemotherapy. In the study, remission was induced by administering imatinib from day 8 to day 62 in combination with cyclophosphamide, daunorubicin, vincristine (VCR), and prednisolone (PSL). Consolidation regimen consisted of an odd course comprising high-dose methotrexate and high-dose cytarabine and an even course with 28 days administration of single-agent imatinib. The consolidation regimens were alternated for four courses each. Maintenance consisting of VCR, PSL, and imatinib was continued for two years after a CR. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was recommended if HLA identical sibling donor was available and was allowed from an alternative donor. A total of 103 newly diagnosed Ph+ALL patients were enrolled in the study between August 2002 and August 2004. Median age of the patients was 45 years (range, 15-64 years), and there were 57 males and 46 females. Median follow-up period was 2.6 years (range, 0.1-5.1 years). A CR was achieved in 100 (97.1%) of the 103 patients and not achieved in a patient in whom imatinib was discontinued because of ileus. There were two early deaths during induction. The probability of overall survival (OS) rate for the entire group at three years was 56.8%. No severe adverse effects were observed. Allo-HSCT was performed in the 1st CR (CR1) in 54 of the 74 CR patients under 55 years of age. Relapse occurred in 18 of 20 patients (90.0%) in whom allo-HSCT was not performed in CR1, but in only seven of the 54 patients (13.0%) who underwent allo-HSCT. At three years, the probability of OS rate for patients under 55 years of age was 75.0% in the transplanted group and 36.4% in the non-transplanted group. Allo-HSCT was performed in CR1 in eight of the 25 patients over 55 years of age. Two were myeloablative and six reduced intensity conditionings. Seven of eight patients who underwent HSCT are still alive in a CR. However, the probability of OS rate at three years in the non-transplanted group was 43.2%. In the group that did not receive allo-HSCT in CR1, age (55< years or 55> years), WBC count, bcr/abl transcript level, bcr/abl transcript type (major or minor), co-expression of myeloid antigens (CD13 and/or CD33), and additional chromosomal abnormalities at diagnosis were not associated with OS. The results demonstrated that the imatinib-combined chemotherapy regimen was effective and feasible. The regimen provided a better chance to receive allo-HSCT which resulted in an excellent outcome. However, relapse still remains a problem, especially in patients who are not candidates for allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Abstract 1058 Poster Board I-80 High-dose ara-C is a basic regimen for the treatment of either relapsed or refractory AML. To improve the outcome of salvage therapy, various combinations of high-dose ara-C have being studied. We herein report the outcome of a phase 2 study of FLAGM. Between 2004 and 2008, JALSG conducted a phase 2 study of FLAGM for relapsed or refractory AML to evaluate its efficacy and toxicity. We thereafter further attempted to predict the response to FLAGM by determining the capability of blast cells to yield the intracellular active metabolite of ara-C: ara-C triphosphate (ara-CTP) in vitro before the start of treatment. JALSG FLAGM enrolled 41 relapsed or refractory AML patients (pts) aged 15-64 yrs (median 52 yrs). The patients were treated with fludarabine 15 mg/m2 twice daily (bid)(d1-4), as well as with ara-C 2g/m2, bid (d1-4), G-CSF 300 μg/m2 (d1-4), and mitoxantrone 10 mg/m2 (d3-5). Six pts showed primary refractory AML. 21 pts relapsed within 12 months after the 1st complete remission (CR), whereas 11 pts suffered their 1st relapse later than 12 months thereafter. Only 1 pt with a 2nd relapse was enrolled. Two pts showed refractory AML after relapse. The diagnoses according to the FAB criteria were as follows: M0 - 3 pts, M1 - 7 pts, M2 – 21 pts, M4 - 8 pts, M5 - 2 pts. Performance status: ECOG 0, 1 and 2 in 76%, 22% and 2% of the pts, respectively. Eight pts had good-risk cytogenetics, 26 pts had intermediate-risk cytogenetics, 2 pts poor-risk cytogenetics, no metaphases were obtained and no cytogenetics were performed in 5 pts. Twenty-one blast samples were evaluated for the production of ara-CTP. The blasts were incubated in vitro either with or without 10 μM fludarabine nucleoside (F-ara-A) for 3 h, followed by washing in fresh media and then subsequent incubation with 10 μM ara-C for 3 h. The nucleotide pool was extracted from each sample and applied to HPLC to measure the ara-CTP production. Following FLAGM, 28 pts (68%) achieved a CR and 1 pt (2%) a CR with an incomplete platelet count recovery (CRp), thus resulting in an overall response rate of 70%. The CR rate of pts in primary refractory and that of pts in 1st relapse were 50% and 79%, respectively. 83% of the pts with a late relapse (duration of 1st CR >=12 months) and 77% of the pts with an early relapse (duration of 1st CR Disclosures: No relevant conflicts of interest to declare.

[Purpose] To compare the incidence, onset and severity of hepatic toxicities and their ultimate impact on survival after reduced-intensity HCT (RIST) and conventional HCT (CST). [Patients and methods] We retrospectively reviewed the data of 151 patients with AML (n=86), MDS (32) and overt AML (33) who underwent allogeneic HCT after either RIST (n=78) or CST (73) between Jan, 2000, and Oct, 2004. Conditioning regimens for RIST included busulfan plus fludarabine or cladribine with or without 2–4 Gy TBI, and those for CST included cyclophosphamide with 12 Gy TBI or busulfan. The median age of the patients was 55 years (range, 15–67) for the RIST group and 37 years (1–57) for the CST group. Patients received marrow (n=64), G-CSF-mobilized PBSC (77) or cord blood (10) from either an HLA-matched relative (59), a mismatched relative (19) or an unrelated volunteer (73). The severity of liver toxicities was assessed by maximal measurements of total bilirubin, ALP, AST and ALT according to CTCAE v.3. Causes of hepatic toxicities were assessed basically by the clinical presentation and, in selected cases, by liver biopsy and autopsy results. The median follow-up of surviving patients was 1,401 days (598–2,317). [Results] The incidences of grade 3–4 hepatic toxicities in the RIST and CST groups were, respectively, 38% vs 38% at day 30, and 60% vs 56% at day 100. The timing of maximum hepatic toxicity was also similar between the two groups. However, the incidence of grade 3–4 hyperbilirubinemia was higher in the RIST group after day 60, probably due to the late onset of acute GVHD after the early tapering of immunosuppression in this group. In contrast, there was a trend for more grade 3–4 abnormal transaminase levels in the CST group than in the RIST group, probably due to the toxicities of drugs including myeloablative conditioning. The causes of grade 3–4 hepatic toxicities were GVHD (n=27), drugs (27), conditioning regimen (7), VOD (3), and others (13). Among the 68 RIST and 66 CST patients who survived more than 100 days after HCT, overall survival (OS) was significantly worse in those who developed grade 3–4 hyperbilirubinemia during the first 100 days after HCT than in those who did not (Figure; 1-year OS, 38% vs 76% after RIST and 33% vs 79% after CST). In a multivariate ananysis, grade 3–4 hyperbilirubinemia (HR 1.8 [95% CI, 1.2–2.7], p=0.02) and non-remission status before HCT (HR 2.5 [95% CI, 1.5–4.4], p=0.0001) were associated with an increased risk of mortality. However, grade 3–4 abnormality in ALP and AST/ALT during the first 100 days after HCT was not a significant risk factor for mortality. [Conclusions] Contrary to our expectations, our data showed a similar incidence of hepatic toxicities between the RIST and CST groups. This may be due to an older age population, the use of busulfan in the conditioning regimen, and the rapid tapering of immunosuppression in the RIST group. The outcome of patients who developed severe grade 3–4 hyperbilirubinemia was poor regardless of the conditioning regimen. Figure Figure

Background: Although acute GVHD (aGVHD) is a significant cause of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (HSCT), its impact on outcomes has not been fully evaluated in older patients who receive reduced-intensity HSCT (RIST). Patients and Methods: We retrospectively surveyed the data of 334 patients (median age, 46 years: range, 1–68) with hematological malignancies who underwent allogeneic HSCT between January, 2000, and June, 2005. The diagnoses included AML/MDS (n=164), CML (n=31), ALL (n=39), lymphoma (n=95), and other malignancies (n=5). Sixty-nine patients (21%) had low- and 265 (79%) had high-risk disease. Patients were conditioned with a fludarabine-based reduced-intensity (n=169) or conventional myeloablative regimen (n=165). Patients received bone marrow (n=131), G-CSF-mobilized PBSC (n=173) or cord blood (n=30) from either an HLA-matched relative (n=138), a mismatched relative (n=41) or an unrelated volunteer (n=155). The median follow-up of surviving patients was 1,210 days (range, 346–2,317). Results: Grade II-IV aGVHD occurred in 146 patients (44%; grade II, n=82; grade III, n=43; grade IV, n=21), 73 patients each in the myeloablative and RIST groups, and the timing was before disease progression or relapse in 144 patients. The incidence of NRM in these 144 patients with aGVHD was 41% (59/144), while this was 29% in the overall 334 patients. The causes of NRM included GVHD (n=22), infections with (n=19) or without (n=9) GVHD, and others (n=9). Hence, aGVHD was the primary cause of NRM. Among these 144 patients with grade II-IV aGVHD, 121 (84%) received ≥1 mg/kg/day of methylprednisone as a primary treatment for GVHD, and 19 of the 121 patients (16%) required additional treatment including switching to tacrolimus (n=11), MMF (n=9), or ATG (n=4). Subsequently, 93 patients (65%) developed chronic extensive GVHD (46 progressive and 47 quiescent). The 2-year probability of NRM after the development of aGVHD was higher as the patient age increased; i.e. 29% for Conclusions: Our data suggest that older patient age is a significant risk factor for NRM after the development of aGVHD, regardless of the type of conditioning regimen. The development of a careful management of aGVHD, focusing on older patients should be the subject of intense clinical research. Figure Figure

The commitment and differentiation of hematopoietic progenitors are thought to be associated with the restriction of transcriptional accessibility for genes of unselected cell-lineage. Histone acetylation-mediated remodeling of chromatin structures is known to regulate transcriptional activity of genes, and the acetylation level of histones is controlled by the balance between opposing activities of histone acetyltransferases and histone deacetylases (HDACs). In this study, we investigated the role of HDAC activity in the proliferation and differentiation of erythroid and granulocytic precursors, using a HDAC inhibitor FK228 [depsipeptide]. CD36+ erythroid precursors were generated from cord blood CD34+ cells with SCF, flt-3 ligand, TPO, IL-3, IL-6 [5GF], and EPO. These precursors expressed low or undetectable level of glycophorin A (GPA) but mostly expressed CD45. When these precursors were cultured with EPO alone in the presence of FK228 (0.4~0.5 ng/ml), the resulting cells expressed GPA at a low level in comparison with the cells cultured without FK228, and a large population of the GPA+ cells remained to be CD45+. On the other hand, most of cells in cultures without FK228 expressed a high level of GPA and a majority of the GPA+ cells were negative for CD45. These data suggest that FK228 represses the EPO-dependent differentiation of erythroid precursors. The retardation of differentiation due to FK228 was, however, completely recovered when cells exposed to FK228 were incubated for additional 5 days without FK228. At these concentrations, FK228 did not affect the proliferation of erythroid precursors. Granulocytic precursors were also prepared from CD34+ cells with 5GF and G-CSF. In culture with G-CSF, FK228 did not affect the differentiation of granulocytic precursors but did inhibit their proliferation in a dose-dependent fashion. Histone acetylation induced by FK228 in precursors was confirmed using anti-acetylated histone antibodies. These data demonstrate a distinct and regulatory role for HDAC activity in the proliferation and differentiation of hematopoietic precursors.