ALBERT

Description: Abstract 1743 Poster Board I-769 Background Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. Aims Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. Results Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. Conclusions Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Description: Abstract 3277 Poster Board III-1 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells. Disclosures: Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

Description: Mechanical tension is a critical determinant of vascular cell growth, differentiation, apoptosis, migration and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin αvβ3 in NIH3T3 cells by using conformation specific antibody WOW-1. LM609, an antibody against αvβ3 integrin that is insensitive to integrin conformation, showed no change in binding after stretch, excluding changes in surface expression. Stretch also induced an increase of LIBS-6 binding, evidence that conformational activation leads to new binding of integrins to ECM. PI3K and Akt are both rapidly activated by strain, and PI3K inhibition decreases integrin activation. These data strongly suggest that PI3K mediates αvβ3 integrin activation. The blocking anti-fibronectin antibody 16G3 strongly inhibited the stretch-induced activation of JNK under conditions where existing adhesions did not appear to be disrupted, indicating that new integrin-ECM binding is required for JNK activation. These data define a pathway by which early activation of PI3K, through induction of integrin activation and ECM binding, stimulates a cytoplasmic signaling pathway implicated in cellular responses.

Description: Abstract 4334 BACKGROUD We designed a multicenter study (JALSG ALL 97) including an intensified consolidation program with dose-escalated doxorubicin (DOX) in order to improve outcome in adults with acute lymphoblastic leukemia (ALL) in pre-imatinib era. We reported here the efficacy and prognostic factors of mainly Philadelphia chromosome (Ph)-negative patients. METHODS From May 1997 to December 2001, patients (age ranges 15 - 64 years) with previously untreated ALL (excluding mature B-cell ALL) were consecutively registered in this study. We modified the standard induction program with five drugs; vincristine (VCR), daunorubicin, cyclophosphamide, prednisolone (PSL) and L-asparaginase and the maintenance program with daily 6-mercaptopurine, weekly methotrexate (MTX) and monthly pulses of VCR and PSL used in CALGB 8811 study. Consolidation therapy included eight courses featuring dose-intensified DOX and intermediate-dose MTX. The total dose of DOX in consolidation phase was 330 mg/m2. For patients with Ph or t(4;11), allogeneic stem cell transplantation (HSCT) was recommended during their first complete remission (CR), if donors were available; whereas for patients without Ph or t(4;11) there was no criteria for choosing HSCT. The 5-year overall survival (OS), the 5-year disease-free survival (DFS), and the prognostic factors were evaluated. RESULTS There were 404 eligible patients (median age, 38 years), of whom 256 were Ph-negative and 116 were Ph-positive. Of the eligible patients, 298 patients (74%) achieved CR. With a median follow-up time of 5.8 years, the estimated 5-year OS rate was 32% (95%CI: 27.1-36.9), and the 5-year DFS was 33% (95%CI: 26.8 - 38.2). The CR rates in Ph-negative and Ph-positive patients were 81% (n=208) and 56% (n=65), respectively. The 5-year OS in Ph-negative and Ph-positive patients were 39% and 15%, respectively. In Ph-negative patients, multivariate Cox analysis showed that older age, PS and WBC count were the independent prognostic factors for OS. The 5-year OS rates for patients younger than 35 years and a WBC count less than 30 × 109/L (risk group 1), for patients younger than 35 years and a WBC count above 30 × 109/L (risk group 2), for patients older than 35 years and a WBC count less than 30 × 109/L (risk group 3), and for patients older than 35 years and a WBC count above 30 × 109/L (risk group 4), were 51%, 29%, 33%, and 27%, respectively (P=0.0005). Of the 208 Ph-negative patients who achieved CR, 60 patients (29%) were underwent allogeneic-HSCT during their first CR (37 from a related donor and 23 from an unrelated donor), resulting that 8 (13%) died in remission, 16 (27%) relapsed, and 36 (60%) remained in continuous CR. The 5-year OS rate for the 60 patients was 63 %. Among them, the 5-year OS rates for the 31 patients of the risk group 1 (standard risk group) and for other 29 patients (high risk group) were 73% and 54%, respectively. Among 148 patients who did not receive allogeneic-HSCT during first CR, six (4 %) died in remission, 105 (71%) relapsed, and 37 (25%) remained in continuous CR. The 5-year OS rates for the 148 patients, for patients with standard risk, and for patients with high risk were 37%, 44% and 33%, respectively. CONCLUSION Result of this study was in the range of those reported by most large cooperative groups, but showed little improvement of adult Ph-negative ALL therapy. The prognostic factors for long term outcome of Ph-negative patients were similar to those in previous reported. This study also suggested that allogeneic-HSCT for Ph-negative patients in first CR might have contributed to the improvement of the outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Rituximab is a murine/human chimeric-anti-CD20 monoclonal antibody that has become a key molecular-targeting drug for CD20-positive B-cell lymphomas. Although combination chemotherapy with rituximab has provided remarkably favorable results for CD20-positive B-cell lymphoma patients, acquired resistance to rituximab has become a considerable problem. Several mechanisms of resistance have been predicted, but the clinical significance of those mechanisms has remained unclear. Previously, at the last ASH Meeting, we showed that down-regulation of CD20 protein expression after using rituximab-containing chemotherapy is one of the critical mechanisms of rituximab resistance, and some epigenetic mechanisms, in part, were related to the aberrant down-regulation of MS4A1 (CD20) gene transcription. On the other hand, we have also found that some patients show resistance to rituximab even in the presence of CD20 protein expression. With this backgrounds in mind, we here investigated he relationship between CD20 protein expression condition and the responsiveness to treatment with rituximab. First, using B-cell lymphoma cell lines and primary lymphoma cells obtained from B-cell lymphoma patients, the CD20 protein expression condition was confirmed by immuno-histochemistry, flow cytometry (FCM), and immunoblotting (IB). In IB analysis, two different sizes of CD20 protein, upper (~37 kDa) and lower (~35 kDa), were confirmed. In vitro phosphatase assay suggested that the upper band is a phosphorylated band of the wild-type CD20 protein. The intensity of those two bands was measured, and the upper/lower (U/L) ratio was calculated. Interestingly, the U/L ratio in diffuse large B-cell lymphoma (DLBCL) cells was significantly lower (range; 0.1 to 0.3) than that of grade 1 and 2 follicular lymphoma (FL) and B-chronic lymphocytic leukemia (B-CLL) cells (0.3 to 0.9 and 0.5 to 0.9, respectively). Next, we tried to analyze the rituximab-inducing complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activities using in vitro chrome-releasing assay. Generally, the CDC/ADCC activity in the CD20(−) cells was significantly lower than that of CD20(+) rituximab-sensitive cells. In contrast, the CDC/ADCC activity in CD20(+) cells varied considerably, with the result that the protein expression level measured by FCM and IB was not always proportional to rituximab sensitivity. In one FL patient showing clinically rituximab resistance in spite of the CD20(+) phenotype, CDC/ADCC activity in vitro was also significantly lower than that of rituximab-sensitive control cells, and the U/L ratio was remarkably high (~1.7) compared to CD20(+) DLBCL and FL cells obtained from rituximab-sensitive patents. Our findings suggest that some kind of B-cell lymphoma, e.g. DLBCL, can be differently classified by CD20 protein expression pattern according to the U/L ratio, and the responsiveness to rituximab may not be determined only by the whole CD20 protein expression level. Furthermore, our data may also suggest a possibility that the responsiveness to rituximab is, in part, modulated by post-translational modification of CD20 protein in vivo.

Description: Constitutive activation of FLT3 kinase is associated with poor prognosis in AML and is present in 30–40% of AML patients. KW-2449 is a multi-kinase inhibitor against FLT3, FGFR1, Abl and Abl-T315, tyrosine kinases with IC50 values of 0.007, 0.036, 0.014 and 0.004 μmol/L, respectively. While it has inhibitory activity against c-Src, JAK2 and c-Kit tyrosine kinases with IC50 values between 0.15–0.40 μmol/L, it has little effect on kinase activity of PDGFR, Fms, IGF1R, and EGFR at a concentration of 1.0 μmol/L. KW-2449 uniquely inhibits Aurora A serine threonine kinase with IC50 of 0.048 μmol/L. Since KW-2449 possesses potent FLT3 as well as Aurora inhibition activity, we studied its possible mode of actions for anti-leukemic effects in both in FLT3 constitutively activated and FLT3 wild type leukemia cell lines. In FLT3-ITD (+) MOLM-13 cells, the exposure of KW-2449 induced a concentration-dependent reduction of phosphorylated FLT3 (P-FLT3) and STAT5 (P-STAT5), a key downstream signal molecules of FLT3. In MOLM-13 cells, KW-2449 also induced G1 arrest and apoptosis over the GI50 value (0.01 - 0.02 μmol/L). In addition, KW-2449 showed potent growth inhibitory activity against 32D cells transfected with FLT3-ITD, FLT3-D835Y or WTFLT3 plus FL, with GI50 values below 0.05 μmol/L. It also inhibited the growth of FLT3 wild type human leukemia RS4;11 cells giving the GI50 value of 0.23 μmol/L. Exposure of RS4;11 cells to KW-2449 also induced the reduction of phosphorylated Histone H3 which is a key downstream signal for Aurora kinase, as well as G2/M arrest and apoptosis over the GI50 value. These data provide additional evidence that Aurora inhibition may play a critical role in the anti-proliferative effects of KW-2449 in FLT3 wild type leukemia. To assess the anti-leukemia activity of KW-2449 in vivo, the SCID mice subcutaneously inoculated with MOLM-13 leukemia were treated with oral KW-2449 bid for 14 days. Dose- dependent tumor growth inhibitory activity was observed at doses ranging from 2.5 to 20 mg/kg. Treatment with 20 mg/kg bid induced complete remission of inoculated tumour in all mice in the cohort without causing significant body weight loss. A PK/PD study using this model revealed significant decreases of P-FLT3 and P-STAT5 in tumors 4 to 8 hours after KW-2449 treatment at 20 mg/kg. In addition, oral administration of 20 mg/kg of KW-2449 showed the survival-prolongation effect in two xenograft models in which the mice were intravenously inoculated with MOLM-13 or 32D/FLT3-D835Y. Furthermore, anti-proliferative activity of KW-2449 was examined in primary samples from AML patients with FLT3 mutation or wildtype. The concentration-dependent growth inhibitory effects that were correlated with P-FLT3 and P-STAT5 reduction were observed in the primary leukemia cells with FLT3 activating mutation. Since KW-2449 shows a potent and unique kinase inhibition profile, it is expected that it could be effective not only in the treatment of AML with FLT3 mutations, but also in FLT3 wild-type AML, imatinib-resistant chronic myeloid leukemia (CML), ALL, and other hematological malignancies. KW-2449 is being investigated in a Phase I, open label, single agent, dose-escalation, multi-center study in patients with relapsed and refractory AML, MDS, CML and ALL.

Description: KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. In this study, we examined its possible modes of action for antileukemic effects on FLT3-activated, FLT3 wild-type, or imatinib-resistant leukemia cells. KW-2449 showed the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G1 arrest, and apoptosis. Oral administration of KW-2449 showed dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, it induced the reduction of phosphorylated histone H3, G2/M arrest, and apoptosis. In imatinib-resistant leukemia, KW-2449 contributed to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. Furthermore, the antiproliferative activity of KW-2449 was confirmed in primary samples from AML and imatinib-resistant patients. The inhibitory activity of KW-2449 is not affected by the presence of human plasma protein, such as α1-acid glycoprotein. These results indicate KW-2449 has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. Our studies indicate KW-2449 has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as imatinib-resistant mutations.

Description: Background: Invasive fungal infections (IFIs) are of serious concern in the management of immunocompromised patients (pts) with hematological disorders. Empiric antifungal therapy is recommended for neutropenic pts with persistent fever, because treatment after confirmation of fungal infection often produces poor outcomes. Micafungin (MCFG), one of the echinocandin families, was launched first in Japan in 2002, and has now been approved in more than 11 countries and areas including the USA and the EU. Although the efficacy and safety of MCFG against both Candida and Aspergillus infections has been shown in many clinical trials, there are few clinical study reports on the empiric therapy of a suspected fungal infection. Here, we report the multi-center study results of MCFG for the empiric antifungal therapy, which were conducted from April 2005 to September 2006 in Japan. Objective: This prospective study was performed to clarify the efficacy and safety of MCFG for the empirical antifungal therapy on suspected fungal infection in pts with hematological disorders and neutropenia. Methods: Study design: A multiple-center, open, uncontrolled study. The investigator registered pts with neutropenia (〈 1,000/μl) who met the following criteria to the Subject Registration Center. Suspected fungal infections were divided into two categories: possible fungal infection defined by positive clinical symptoms/findings and serological testing (beta-D-glucan or galactomannan) or diagnostic imaging (chest X-ray or CT scan), refractory fever defined by unexplained persistent fever (an axillary temperature higher than 37.5 °C) after the antibacterial treatment over 2 days and by positive clinical symptoms/findings. IFIs categorized as proven or probable were not included in this study. Efficacy evaluation was performed using an algorithm based on each of the evaluation of clinical symptoms/findings, imaging study findings, and serological tests. Results: 388 pts (M:234, F:154, mean age:57.8 years old) were registered. The mean dosage and duration of treatment with MCFG were 154.6±55.3 mg/day and 14.0±6.9 days, respectively. The main underlying hematological disorders were acute leukemia (61.3%), non-Hodgkin’s lymphoma (18.3%) and myelodysplastic syndrome (10.8%). The number of pts with hematopoietic stem cell transplantation (HSCT) was 76 (19.6%). The clinical response rate (CRR), excluding 4 non-evaluable pts was 63.3% (243/384): 60.1% (89/148) for pts with possible fungal infection and 65.3% (154/236) for pts with refractory fever, respectively. Even in persistent neutropenic pts whose neutrophil counts were 〈 500/μL throughout the treatment with MCFG, the CRR was high enough: 46.9% (61/130). No difference was observed in the CRR among the main underlying hematological disorders. The CRR in pts with HSCT and other conditions were 63.2% (48/76) and 63.3% (195/308), respectively. Drug-related adverse events (DAEs) were observed in 16.8% (65/388). Serious DAEs such as elevation of serum bilirubin and renal dysfunction was observed in 0.52% (2/388). Conclusion: MCFG was confirmed to have high clinical efficacy and be safe for the treatment of suspected fungal infection in pts with hematological disorders and neutropenia.

Description: A randomized study had been performed between December 2001 and December 2005 to assess the optimal post remission therapy for adult AML in the first CR. The updated results are here presented, after a median follow-up of 48 months. JALSG AML201 enrolled 1064 previously untreated AML patients (pts) aged 15–64 yrs. The induction therapy consisted of cytarabine (Ara-C 100 mg/m2 day1–7) and idarubicin (IDR 12 mg/m2 day1–3) (arm A) or cytarabine (100 mg/m2 day1–7) and daunorubicin (DNR 50 mg/m2 day1–5) (arm B). If the patients did not achieve remission after the first induction therapy, then the same therapy was given once more. Pts were categorized into good, intermediate or poor risk groups by risk factors based on the criteria established in previous JALSG AML studies (Miyawaki et al. Cancer 2005). All CR pts were stratified according to the induction, the number of courses of induction, age and karyotype and were randomly assigned to the high dose Ara-C (HDAC) post remission regimen (arm C) or the conventional JALSG post remission regimen (arm D). Arm C: the three courses of HDAC which consisted of Ara-C 2.0 g/m2 q12h day1–5, arm D: the first course consisted of Ara-C 200 mg/m2 day1–5+ mitoxantrone (MIT) 7 mg/m2 day1–3, 2) Ara-C 200 mg/m2 day1–5+ DNR 50 mg/m2 day1–3, 3) Ara-C 200 mg/m2 day1–5+ aclarubicin (ACR) 20 mg/m2 day15, 4) Ara-C 200 mg/m2 day1–5+ etoposide (ETP) 100 mg/m2 day1–5 + vincristine (VCR) 0.8 mg/m2 day 8 + vindesine (VDS) 2 mg/m2 day10. Results: Of the 1064 pts registered, 1057 pts (median age: 47 years) were evaluable. 823 pts (78%) achieved CR after one or two courses of induction therapy. Of the 823 pts in CR, 781 pts were assigned to arm C (n=389) or arm D (n=392). The 5-year OS rate of arm C was 57.8% while that of arm D was 55.9% (p=0.96). The 5-year RFS rate of the CR pts was 42.7% in arm C and 38.9% in arm D (p=0.73). Among the good risk group (n=155), the 5-year OS rate of arm C was 69.9% while that of arm D was 80.5 % (p=0.11), and the 5-year RFS rate of arm C was 54.5% while that of arm D was 55.7% (p=0.53). Among the intermediate risk group (n=439), the 5-year OS rate of arm C was 50.9% while that of arm D was 48.5% (p=0.59), and the 5-year RFS rate of arm C was 41.5% while that of arm D was 36.5% (p=0.50). Among the poor risk group (n=49), the 5-year OS rate of arm C was 12.9% while that of arm D was 17.2% (p=0.58), and the 5-year RFS rate of arm C was 14.3% while that of arm D was 15.5% (p=0.78). In the CBF leukemia group (n=218), the 5-year OS rate of arm C was 75.0% while that of arm D was 65.8% (p=0.17), and the 5-year RFS rate of arm C was 56.5% while that of arm D was 38.7% (p=0.05). Among the young group (=50 yrs) (n=314), the 5-year OS rate of arm C was 51.3% while that of arm D was 40.1% (p=0.16), and the 5-year RFS rate of arm C was 40.0% while that of arm D was 28.1% (p=0.23). After all of consolidation, the lowest WBC count and the duration of neutropenia in arm C were significantly lower and longer than those in arm D. There was a higher rate of documented infection in arm C (20.9%) than in arm D (14.5%) (p〈 0.001). Conclusion: The conventional post remission therapeutic regimen established by JALSG consisting of 4 courses of consolidation was found to be as effective as the three courses of HDAC therapy. HDAC therapy produced a slightly positive effect on RFS in only the CBF leukemia group.