ALBERT

Description: Aim: To make clear the feasibility of Ifosfamide/Etoposide (IE) regimen combined with granulocyte colony-stimulating factor (G-CSF) for the collection of autologous peripheral blood stem cells (APBSC), we evaluated the number of the collected CD34 positive cells, and the regimen-related toxicities. Patients’ characteristics and Methods: 19 Patients (male 12, female 7) were received APBSC harvest in our hospitals during Aug. 2000 to Dec. 2003 with age of 54.3 years on average (30–66). Diagnoses were included HD (n=2), NHL (n=13), MM (n=3), and malignant synovioma (n=1). IE regimen was included with Ifomide 2000 mg/m2 (day 1), and Etoposide 200 mg/m2 (day 1–3) (4cases) or 500 mg/m2 (day 1,2) (15 cases). For the prevention of hemorrhagic cystitis patients were administered with Mesna, and NaHCO3 during and after 1 day of the administration of Ifosfamide. G-CSF was administered at the dose of 10 ug/kg (lenograstim) or 300 ug/m2 (filgrastim) when blood absolute neutrophil count came down to 1000/mm3. When the suppression of bone marrow was recovered and blood WBC count came up to 5000/m3, CD34-positive cells were counted in blood, and APBSC was harvested when blood CD34-positive cells were determined above 0.1 %. APBSC was harvested with CS-3000 plus (Fenwall). Result: The median duration from the start of the administration of G-CSF to the finish of APBSC harvest was 6.5 days (4–11). The median number of CD34-positive cells of the harvested was 4.32 x 106/kg (1.10–13.50). All cases were harvested. The toxicities during from the conditioning to the harvest were included with grade 1 headache (2 cases), grade 2 nausea (6 cases), grade 1 bleeding (1 case), grade 1 constipation (1 case), grade 1 fever (1 case), and grade 1 thrombocytopenia (1 case). Conclusion: IE regimen combined with G-CSF was feasible to harvest APBSC on the yield of the collection of the transplantable stem cells, and on the regimen-related toxicities.

Description: Aim: To make clear the feasibility of non-myeloablative conditioning on the adult allogeneic umbilical cord blood transplantation, we compared the result between patients receiving the myeloablative (conventional) transplantation (CT) and the reduced intensity stem cell transplantation (RIST) in point of the engraftment, and the incidence of GVHD. Patients’ characteristics and Methods: Patients were admitted to our hospitals to receive umbilical cord blood transplantation during May 1999 to May 2004. CT group (n=23): Median age was 33 years. Diagnoses were included AML (n=9), ALL(n=7), NHL(n=4), MDS(n=2), CML(n=1). Preparative conditioning regimens were; TBI/Ara-C/CY (n=16), TBI/VP-16/CY (n=4), TBI/CY (n=3). GVHD prophylaxis was; Cyclosporine A (CsA) (n=11), CsA with short term Methotrexate (MTX) (n=10), Tacrolimus with short term MTX (n=2). The transplanted median cell number was 2.40 x 10^7/kg. HLA disparities were; 1AMM (n=5), 2AMM (n=18), 3AMM (n=3). RIST group (n=13): Median age was 54 years. Diagnoses were; AML (n=3), NHL (n=5), HD (n=2), ATL (n=1), CLL (n=1), CMMoL(n=1). Preparative conditioning regimens were; Flu/ L-PAM (n=4), TBI/ Flu/ L-PAM (n=1), TBI/ Flu/ CY (n=3), TBI/ Flu (n=1), TBI/ Flu/ BU (n=4). GVHD prophylaxis was; CsA (n=5), CsA with MMF (n=7), Tacrolimus (n=1). The transplanted median cell number was 2.49 x 10^7/kg. HLA disparities were; identical (n=3), 1AMM (n=2), 2AMM (n=6), 3AMM (n=2). Result: The rate of engraftment failure including conditioning failure, and autologous recovery was 14.8 %in CT and 38.1%in RIST, respectively. The median duration of the engraftment of neutrophils in CT and RIST were 24 days and 19 days, respectively. Patients with Grade II-IV acute GVHD were 15 (65.2%) in CT and 8 (61.5%) in RIST, respectively. The incidence of Grade III-IVacute GvHD was 7 ( 30.4%) and 5(38.4%), respectively. Conclusion: Engraftment in RIST was achieved earlier than CT, however the incidence of engraftment failure was increased in RIST. There were no significant differences on the incidence of acute GVHD between two groups. Further observations, especially long-term disease-free survival are seemed to be very interesting to ascertain RIST on adult umbilical cord blood transplantation.

Description: Background: Cytogenetic abnormalities at diagnosis are recognized as a potent prognostic factor for acute leukemia patients. Among acute myeloid leukemia patients, the prognostic implications of cytogenetic abnormalities have been established for those treated with chemotherapy as well as those undergoing allo-SCT. In the context of Ph-negative ALLpatients, cytogenetic abnormalities at diagnosis clearly stratify the prognosis, whereas it has not been elucidated whether similar prognostic stratification is applicable to allo-SCT recipients. Objective: The aim of this retrospective study was to assess the prognostic impact of cytogenetic abnormalities in adult Ph-negative ALL patients who underwent allo-SCT. Patients and Methods: The study cohort included 373 adult Ph-negative ALL patients aged over 15 years who underwent allo-SCT for the first time between January 2001 and December 2012 at the 23 institutions participating in the Kanto Study Group for Cell Therapy (KSGCT). Patients' clinical data were collected from the KSGCT database. The Institutional Review Board of Gunma University approved the protocol of this study. Karyotypes considered high risk (HR) included t(4;11), t(8;14), low hypodiploidy, and complex (equal or more than five abnormalities), and all other karyotypes were designated standard risk (SR). On this basis, 308 patients (82.6%) were categorized as SR and 65 patients (17.4%) were categorized as HR at diagnosis. Of the 373 patients, 267 underwent allo-SCT in complete remission (CR) (224 in the SR group and 43 in HR group), and 106 in non-CR (84 in the SR group and 22 in HR group). For analysis, the study population was stratified based on disease status at the time of transplant. Almost all patients were conditioned with total body irradiation (TBI)-containing myeloablative conditioning (MAC) regimens prior to transplantation. Overall survival (OS) was defined as the interval from the date of transplantation to the date of death. Non-relapse mortality (NRM) was defined as any death in continuous complete remission (CR). The Fisher's exact test was used for comparison of binary variables. OS and RFS were estimated by the Kaplan-Meier method, and compared using the log-rank test. Cumulative incidences (CI) of relapse and NRM were compared using the stratified Gray test. P 〈 0.05 was considered as statistically significant. Results: [Patients in CR] No significant difference in patient characteristics and transplant procedures was observed between the SR and HR groups. The 5-year OS rates were similar between the SR and HR groups (60.5% vs. 74.1%, respectively; p = 0.225) (Figure 1). Similarly, there were no significant differences in the 5-year CI of relapse and NRM rates between the two groups (relapse: 26.3% vs. 24.8%, respectively; p = 0.498, NRM: 19.6% vs. 10.0%, respectively; p = 0.232). Multivariate analysis for OS identified MAC and TBI-containing regimens, not cytogenetic risk, as significant positive prognostic factors. [Patients in non-CR] No significant difference was observed between the SR and HR groups in terms of patient characteristics or transplant procedures, although there was a female predominance in the HR group. Patients in the SR group had a significantly superior 5-year OS rate compared to the HR group (15.4% vs. 4.5%, respectively; p = 0.022). There was no significant difference in the 5-year CI of relapse between the SR and HR groups (60.3% vs. 50.0%, respectively; p = 0.411), whereas the 5-year CI of NRM in the SR group was significantly lower than that in the HR group (24.8% vs. 45.5%, respectively; p = 0.024). Multivariate analysis revealed cytogenetic risk group as an independent prognostic factor. Conclusion: These findings suggest that adult Ph-negative ALL patients in remission with HR cytogenetic abnormalities have similar transplant outcomes to those in the SR group. Considering the reported equality of the CR rates between the two groups, allo-SCT at an early clinical phase is recommended for HR group patients, reminiscent of Ph-positive ALL patients in the pre-imatinib era. Current transplant procedures do not improve outcomes for patients who are not in remission, especially those with HR cytogenetic abnormalities. Disclosures Usuki: MSD: Other: personal fees, Research Funding; SymBio Pharmaceutical: Other: personal fees, Research Funding; GlaxoSmithKline: Other: personal fees, Research Funding; Shionogi: Other: personal fees; Fujimoto Pharmaceutical: Research Funding; Bristol-Myers Squibb: Other; Takeda Pharmaceutical: Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; Shire: Research Funding; Kyowa Hakko Kirin: Other: personal fees, Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Novartis: Other: personal fees, Research Funding; Boehringer Ingelheim: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Chugai Pharmaceutical: Other: personal fees; Fuji Film RI Pharma: Other: personal fees; Taiho Pharmaceutical: Other: personal fees, Research Funding; Astellas: Research Funding. Nakaseko:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding.

Description: Objective: It has been reported that acute leukemia with the fusion product of MLL-related molecule is determined to be the poor prognosis. To make clear one of its biological characters, we cultured AML blasts with t (11; 19) (q23; p13.1), which produced fusion protein of MLL and ELL, and the morphological changes as well as their DNA, and RNA were examined. Method: Bone marrow cells were obtained from two patients with MLL-ELL fusion gene after obtaining informed consent. There were over 90% blastic cells in bone marrow. Mononuclear cells were obtained with Ficoll (SG. 1077), and were cultured in DMEM with 10% FCS in CO2 incubator. The morphology of the cells was observed for 3 months. Also, DNA was extracted, and MLL-ELL translocation was analyzed with genomic Southern blotting with probe x which contains BamHI-digested 0.9 kb fragment from MLL cDNA. RNA was extracted with GTC method, and 1st strand cDNA was synthesized with oligo-dT primer, and PCR was performed with MLL common primer and ELL antisense primer. The reaction of DNA sequencing was performed with BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and analyzed with ABI Prism 3700 DNA analyzer. Result: After one month of the cultures, fibroblastoid cells were expanded without any blastic cells onto them. Southern analysis, RT-PCR study, and DNA sequence revealed that the fusion cDNA product with MLL and ELL was observed in fibroblastoid cells. Discussion: AML blasts with MLL-ELL fusion gene can convert their morphology into the fibroblastoid cells. The fibroblasts have been reported to be resistant to the chemotherapeutic agents. This morphological conversion may contribute one of the causes of poor prognosis in AML patients with MLL-ELL fusion gene. We now characterize precisely these fibroblastoid cells in point of the expression of the specific proteins, and the functions including the binding and proliferating capability to the non-adherent blastic cells.

Description: Objective: A 25 years-old Japanese male was diagnosed as AML (M5a) with 46, XY, der (1) t(1; 1) (p36; q21). To determine the leukemogenesis in this case, we analyzed the fusion product of this chromosomal translocation. Method: After obtaining informed consent, RNA was extracted from his bone marrow cells (blastic cells were occupied in almost 100%) with GTC method, and poly-A rich RNA was separated with oligo-dT latex particles. 1st strand cDNA was synthesized with oligo-dT primer. Oligo-dA stretch was added with TdT to make 5′ RACE. 1st PCR was performed with the synthetic antisense-1 primers from several candidates including p73, and MEL1, and with QT primer (5′-TGAGCCAGAGTGACGAGGACTCGAGCTCAAGCT17-3′) as a sense primer. 2nd PCR was performed with internal antisense-2 primers from several candidates, and Q0 primer (5′-CCAGTGAGCAGAGTGACG-3′) as the sense primer. The reaction of DNA sequencing was performed with BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and analyzed with ABI Prism 3700 DNA analyzer. Result and Discussion: The fusion cDNA product was obtained, which was consisted of MEL1 and Phosphatase 10. The breakpoint of MEL1 was exon 4, and PR domain of MEL1 was disrupted. This domain is reported to be the key function of MEL1, and when MEL1 without PR domain is expressed, myeloid differentiation is blocked. In this case the fusion of Phosphatase 10 and MEL1 is seemed to be the main cause of leukemogenesis.

Description: Objective: To understand better the mechanism of the maintenance of immature state of stem cells, murine novel cDNA clones are to be isolated by the gene trap method. Method: The trap vector was constructed with En-2 intron sequence followed by splicing acceptor sequence, IRES signal sequence and b-galactosidase cDNA. NeoR gene was also contained without poly-A additional signal. After electroporation into Embryonic stem (Es) cell lines (D3, and E14.1 Köln), neomycin-resistant Es clones were picked up, and were selected in which the expression of b-gal was detected in the undifferentiated condition cultured with leukemia inhibitory factor (LIF), and feeder layer cells by X-gal staining, but were not detected in the differentiated condition with ATRA, and without LIF nor feeder layers. Using 5′ RACE method, the transcripts from the selected clones were identified. One clone was further analyzed. Full-length cDNA was isolated from a library constructed from Es D3 cells, and its characterization was determined. Result and Discussion: one unique full-length cDNA clone was isolated for the further characterization. It was constructed with 2225 nucleotides, and putative 466 amino acids were encoded in a single long open reading frame. The transcript was detected in the undifferentiated Es cells but not in differentiated Es cells nor in various kinds of organs by northern blotting analysis. When this clone was over-expressed in murine 10T1/2 cells, RT-PCR analysis demonstrated the down-regulation of Id-1 gene. These data suggested that the isolated cDNA clone was possibly related to keeping Es cells to be immature multipotent state. We now try to isolate the related cDNA clones which are expressed mainly in the hematopoietic stem cells.