ALBERT

Description: Abstract 1360 Introduction: The tyrosine kinase inhibitor (TKI) imatinib is used as the first-line therapy for newly diagnosed chronic myeloid leukemia (CML). However, some patients fail to respond or become intolerant to imatinib. Nilotinib is a second-generation TKI with higher selectivity and more potent inhibitory effects on BCR-ABL than imatinib. Several studies have shown hematologic and cytogenetic responses to nilotinib in patients with imatinib-resistant or intolerant CML. Purpose: To investigate the safety and efficacy of nilotinib for patients with imatinib-resistant or intolerant, chronic (CP)- or accelerated (AP)-phase CML from the East Japan CML Study Group (EJCML) trial by evaluating molecular responses in terms of the BCR-ABL1 mutational status and plasma trough concentration of nilotinib. Methods: In this multicenter phase II clinical trial, nilotinib (400 mg bid) was administered orally for one year and the molecular responses were monitored by means of the international scale of quantitative PCR (IS-PCR). BCR-ABL1 mutations were analyzed by direct sequencing at the baseline and 12 months or at the time of the event for discontinuation of the treatment (i.e., progressive disease, insufficient effects, or severe adverse events). The plasma trough concentration of nilotinib was measured by high-performance liquid chromatography 3 months after nilotinib administration. Results: From March 2009 through February 2011, 51 patients were registered in this study, and data of 49 patients whose molecular responses were evaluated by the IS-PCR were analyzed (imatinib-resistant CML = 33, imatinib-intolerant CML = 16; CP CML = 46, AP CML = 3). The median follow-up period was 12.0 months (range = 0.1–13.3 months). At 6 and 12 months, the major molecular response (MMR; ≤0.1% IS) rates were 52.5% and 67.6%, respectively, and the complete cytogenetic response (CCyR)-equivalent (≤1.0% IS) rates were 75.0% and 85.3%, respectively. Five types of BCR-ABL1 mutations (M244V, F317L, N358D, F359V, and E459K) were detected in 6 patients (12.2%) at the baseline, but the M244V, N358D, and E459K mutations disappeared after the nilotinib treatment. Acquired BCR-ABL1 mutations (Y253H, I418V, and exon 8/9 35bp insertion) were detected in 3 patients (8.6%) at 12 months or at the time of the event; these patients did not achieve a CCyR or an MMR. No patients showed an acquired mutation of T315I. Most patients except 11 subjects (22.4%) still received the treatment. The reasons for discontinuation were progressive disease in one patient with an F317L mutation, insufficient effects in one patient without any mutation, and adverse events in 9 patients (thrombocytopenia in 5 patients, hyperbilirubinemia in 2 patients, headache in one patient, and heart disease in one patient). Among 30 patients without BCR-ABL1 mutations, the plasma trough concentration of nilotinib was significantly higher in 21 patients with an MMR than in those without an MMR by 12 months (median = 1255.1 ng/mL vs. 372.8 ng/mL, P = 0.0012 by Mann–Whitney U-test; see the figure). The concentration of 761 ng/mL was significantly associated with an MMR by 12 months in a receiver-operating characteristic (ROC) curve analysis of the best sensitivity (76.2%) and specificity (77.8%). Conclusion: The patients with imatinib-resistant or intolerant, CP or AP CML, even those having BCR-ABL1 mutations M244V, N358D, and E459K, achieved an MMR by 12 months of nilotinib treatment. The plasma trough concentration of the drug was related to the MMR by 12 months, and the plasma threshold of nilotinib should be set above 761 ng/mL. These findings suggest that nilotinib shows good efficacy and tolerability in Japanese patients with imatinib-resistant or intolerant, CP or AP CML. (ClicalTrials.gov, UMIN ID 000002201) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4147 Whole-body diffusion-weighted magnetic resonance imaging (DWI-MRI) provides functional information and is able to highlight oncological lesions, but the usefulness has not been established in malignant lymphoma especially for monitoring therapeutic response. Positron emission tomography with fluorine-18 fluorodeoxyglucose (FDG-PET) is a useful imaging producer for tumor staging and therapy monitoring that can visualize active tumor tissue including malignant lymphoma. The spatial resolution of FDG-PET is limited, and low accuracy rates in diabetic patients and those with low grade lymphoma have been reported. We prospectively studied the utility of DWI-MRI with T2 imaging and apparent diffusion coefficient (ADC) for staging and monitoring therapeutic responses in patients with malignant lymphoma compared with FDG-PET/CT. Twenty-eight patients with malignant lymphoma (16 patients with diffuse large B cell lymphoma: DLBCL, 7 with follicular Lymphoma: FL, 3 with aggressive T cell lymphoma: TCL and 2 with Hodgkin lymphoma: HL, including one diabetic patient) received both MRI and FDG-PET examination before (n=28), after 2 courses of chemotherapy (n=25) and one month after the end of chemotherapy (n=9). MRI examination was performed with a 3-Tesla MR system (Signa Excite, Generel Electrics). Whole-body DWI-MRI was performed with echo planar imaging sequence with short T1 inversion recovery (STIR) fat suppression. ADC measurement was performed based on the region of interest (ROI) method. ROI was placed on the lesion showing the highest standardized uptake value (SUV) on FDG-PET/CT scanner (Discovery LS, General Electrics) in each patient, and crucial parameters of the ADC and SUV were compared. Based on staging by PET/CT, 4 patients were clinical stage I, 8 were stage II, 7 were stage III and 9 were stage IV. DWI-MRI findings alone matched PET/CT in 22 patients (79%), whereas these findings combined with T2 imaging increased match in 26 patients (93%). Regarding the early response to chemotherapy, 19 of 25 patients (76%) were considered to show CR on PET/CT and the DWI findings matched PET/CT 23 patients (92%). To evaluate the final response after chemotherapy, 7 of 9 patients (78%) were considered to show CR on PET/CT and the DWI findings matched PET/CT in 8 of 9 patients (89%). Of these nine, one patient with DLBCL who did not show a match was a false positive on PET/CT. In all patients with TCL and HL, the DWI-MRI findings combined with T2 imaging matched PET/CT findings for staging and therapeutic response. Interestingly, the ADC values on DWI-MRI did not differ between DLBCL and FL (0.77 +/− 0.23 and 0.70 +/− 0.08, p=0.99, mean +/− SD respectively), whereas the SUV values of DLBCL on PET/CT were higher than those of FL (14.5 +/− 6.97 and 6.09 +/− 2.54, p 〈 0.0005, mean +/− SD respectively), suggesting the DWI-MRI could detect the lymphoma lesion more accurately than PET/CT in patients with indolent lymphoma such as FL. We conclude that whole-body DWI-MRI combined with T2 imaging and ADC analysis could be promising sensitive method for staging and therapeutic response evaluation in patients with malignant lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Background Invasive fungal infections (IFIs) are of great concern after allogeneic hematopoietic stem cell transplantation (HSCT), the risk of which is considered to be particularly prominent among cord blood transplantation (CBT) recipients. Patients and Methods We retrospectively analysed the records of 749 adult patients who underwent CBT or unrelated bone marrow transplantation (uBMT) for the first time at the Toranomon Hospital between 2002 and 2012, and who had neither prior history nor suspicious findings of IFIs. As prophylaxis for IFIs, fluconazole (FLCZ) or itraconazole (ITCZ) capsules were conventionally used until around 2006, which were then changed to newer mold-active agents including ITCZ oral solution, voriconazole or micafungin after their approval in Japan, the choice of which was subjected to physician's discretion. Results Engraftment achieved in 418 CBT patients and 198 uBMT patients with a significantly longer neutropenic period in CBT patients (median 20 days vs 18 days, P

Description: Objectives Fever during neutropenia occurs in 〉 90% and 80% of allogeneic and autologous hematopoietic stem cell transplantation (HSCT) recipients, respectively. Current guidelines recommend the prophylaxis with fluoroquinolones (FQs) in HSCT patients. Although there is evidence that antibiotic prophylaxis improve clinical outcome in patients with chemotherapy-induced neutropenia, prophylactic antibiotic therapy has not been thoroughly evaluated in HSCT recipients. Therefore, we performed a meta-analysis to evaluate the impact of systemic antibiotic prophylaxis in HSCT recipients on mortality, incidence of infection and related adverse events. Data sources We identified reports that were not restricted to those in English and not restricted to published trials through PubMed, the Cochrane Library, and references of identified studies. Review Methods We included prospective, randomized studies on systemic antibiotic prophylaxis in HSCT recipients. The outcome measures included the all-cause mortality, infection-related mortality, febrile episodes, incidence of clinically or microbiologically documented infection, bacteremia, or related adverse events. The summarized odds ratios (ORs) were calculated using the Mantel–Haenszel method and the DerSimonian–Laird method. Results Seventeen trials with 1453 patients (842 autologous and 407 allogeneic HSCT recipients) were included. The percentage of autologous and allogeneic HSCT recipients was not specified in 2 trials. Systemic antibiotic prophylaxis was compared with placebo or no prophylaxis in 10 trials and with non-absorbable antibiotic in 2 trials, respectively. Systemic antibiotics other than FQs were evaluated in five out of these 12 trials. Four trials evaluated the effect of addition of antibiotics for gram positive bacteria to FQs. Remaining 1 trial compared the two different systemic antibiotic regimens, FQs versus trimethoprim sulfamethoxazole. As a result, systemic antibiotic prophylaxis reduced the incidence of febrile episodes (OR 0.16; 95 percent confidence interval [CI], 0.09-0.30), clinically or microbiologically documented infection (OR 0.41; 95% CI 0.30-0.57) and bacteremia (OR 0.37; 95% CI 0.26-0.53) without the significant effect on all-cause mortality or infection-related mortality (OR 0.89; 95% CI 0.48-1.66, OR 1.37; 95% CI 0.50-3.76, respectively). Impact of prophylaxis with FQs on mortality was inconclusive because of small number of clinical trials evaluated. Adverse events increased in patients with systemic antibiotic prophylaxis compared to controls (OR 3.32; 95% CI 1.45-7.63). In meta-regression, percentage of allogeneic HSCT recipients was not associated with each outcome measure. With regard to the comparison between different prophylactic regimens, addition of antibiotics for gram positive bacteria to FQs decreased the incidence of bacteremia (OR 0.44; 0.24-0.80) without significant effects on all-cause mortality, infection related death and febrile episodes. There was not significant, but consistent decrease in clinically or microbiologically documented infection (OR 0.55; 95% CI 0.30-1.01). There was significant increase of adverse events in patients receiving addition of antibiotics for gram positive bacteria to FQs (OR 6.65; 95% CI 2.15-20.54). Conclusions Systemic antibiotic prophylaxis successfully reduced the incidence of infection in HSCT recipients. However, there was no significant impact on mortality. Impact of prophylaxis with FQs on mortality in HSCT recipients was inconclusive because of small number of trials evaluated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2007 Background: High-dose cyclophosphamide (HD-CY) + granulocyte-colony stimulating factor (G-CSF) and G-CSF alone have been used to mobilize hematopoietic stem cells (HSCs) for autologous SC transplantation (ASCT) in multiple myeloma (MM). However, which regimen is better is unknown; anti-myeloma effects of HD-CY + G-CSF have not been established. From January 1999 to June 2009, we administered HD-CY+G-CSF but changed to G-CSF alone during July 2009–December 2010. We retrospectively assessed HSC collection efficacy, complications, and anti-myeloma effects of these regimens. Patients and methods: We analyzed 147 MM patients from whom HSCs were to be collected at our institute. For mobilization, 115 patients were administered HD-CY (4 g/m2)+G-CSF (600 mg/body filgrastim or 500 mg/body lenograstim) and 32 were administered G-CSF alone (same dose as HD-CY). Here, 17 patients received therapeutic intervention between mobilization and transplantation without disease progression (PD). To avoid the patient outcome effect, we defined event- and progression-free survivals (EFS and PFS). EFS was defined as PD, death, or therapeutic intervention without PD. PFS was defined as PD or death, where therapeutic intervention without PD was used as a censor. Both were calculated from the start of mobilization. For analyzing response by mobilization, patients receiving therapeutic intervention without PD were excluded. Response was evaluated in those not receiving therapeutic intervention without PD or in whom response could not be evaluated before ASCT. Thalidomide was administered as maintenance therapy to 14 and 6 patients in the HD-CY+G-CSF and G-CSF groups after ASCT. Thalidomide administration was used as a censor. Results: Vincristine, doxorubicin, and dexamethasone (VAD) and HD dexamethasone (HDD) therapies were administered as induction therapy (VAD for 117, HDD for 2, and both for 11). New (bortezomib or thalidomide) and alkylating agents were administered to 7 and 13 patients, respectively. Before mobilization, 26 patients received radiotherapy; none were administered lenalidomide. No statistical difference was seen in baseline characteristics (Durie-Salmon stage, International staging system, interval from diagnosis to mobilization, disease control, and previous therapies) between both groups. However, patients mobilized by G-CSF alone were significantly older. Among 147 patients, 121 underwent planned ASCT. Of the 17 receiving therapeutic intervention without PD, 13 and 4 belonged to the HD-CY+G-CSF and G-CSF groups, respectively. More than 2 × 106 CD34-positive cells/kg were collected from 93% and 75% patients in the HD-CY+G-CSF and G-CSF (p = 0.0079) groups, respectively. More than 4 × 106 CD34-positive cells/kg were collected from 84% and 69% in the HD-CY+G-CSF and G-CSF (p = 0.07). Mean HSC count was 11.4 × 106/kg in the HD-CY+G-CSF group and 4.5 × 106/kg in the G-CSF group (p = 0.0007). Among patients receiving HD-CY+G-CSF, 66% were treated with intravenous antibiotics; 3 suffered cardiac shock and 2 septic shock. However, among those receiving G-CSF alone, no severe complications were seen. Median hospitalization days were 21 and 8 for the HD-CY+G-CSF and G-CSF groups, respectively (p 〈 0.0001). In the HD-CY+G-CSF group, 16% improved in disease control before ASCT, 71% showed no change, and 13% progressed. However, no patient improved, 63% showed no change, and 27% progressed in the G-CSF group (p = 0.015). Median EFS was 25 months in the HD-CY+G-CSF group and 13 in the G-CSF group (fig 1, p value of log-rank test = 0.012). Median PFS was 28 months in the HD-CY+G-CSF group and 15 in the G-CSF group (fig 2, p value of log-rank test = 0.011). Median overall survival did not differ significantly. Conclusion: Regarding the safety and duration of hospitalization, G-CSF alone may be safer and beneficial. However, HD-CY+G-CSF was more effective as a mobilization regimen and showed higher anti-myeloma effects than G-CSF alone. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1684 Imatinib masylate (IM) induces sustained molecular remissions in patients with chronic myeloid leukemia (CML) but a life-long therapy is required for most of these patients. In STIM study, Mahon et al. reported that among patients with a complete molecular response (CMR) lasting at least two years, the CMR was sustained in 41% after discontinuation of IM. German study group showed that treatment with Interferon-alpha (IFN) enables IM discontinuation in most patients after prior IM/IFN combination therapy. We previously reported that CD8+ memory T cells showed significant predominance over naive T cells in the patients with sustained therapy-free major molecular response (MMR), all of that had previously received IFN [1]. We have been performing a phase 2 study of treatment discontinuation after the drug change from IM to IFN (Japanese Imatinib Stop And Interferon Study; JISAS). Since the aim of this study is to confirm the finding that IFN is able to maintain MMR induced by IM after its discontinuation, we present here the result of the interim analysis. Patients Patients with a confirmed diagnosis of CML in 1st chronic phase (CP1) as well as in CMR (undetectable BCR-ABL transcript) following over 2 years of MMR on IM were enrolled in this pilot study after obtaining the written informed consent. All the eligible patients were recruited from September 2009 to December 2011 whether or not any therapeutic drug had been given before IM. Evaluation and response criteria Since a conversion factor for International scale was not available in Japan until recently, for entry to the study, BCR-ABL transcripts at a level equal to or below 100 copy/mg RNA in a real-time quantitative-polymerase chain reaction (RQ-PCR) assay or equal to or below 50 copy/assay in a highly sensitive transcription-mediated amplification (TMA) method were defined as MMR. CMR was defined as detection of no BCR-ABL transcript in RQ-PCR assay, nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay, or TMA. During the study, molecular response was assessed at the baseline and every month thereafter, by determining the BCR-ABL to ABL mRNA transcript ratio isolated from the peripheral blood using RQ-PCR and RT-PCR. The BCR-ABL to ABL transcript ratio at a level below 0.001 was defined as MMR and no detectable BCR-ABL transcript in RT-PCR was defined as CMR. Molecular relapse defined as a loss of MMR was taken into account if confirmed in 2 successive assessments. Study design and treatment Administration of IFN is started at a dose of 3 million units 2–5 times per week within 4 weeks after IM discontinuation. In case of molecular relapse, IM was resumed at 400 mg daily. Statistical analysis Non-parametric values or numbers were compared between the two groups using the Mann-Whitney test. Relapse-free survival was estimated using the Kaplan-Meier method. Results Fifteen patients were enrolled from September 2009 to December 2011. Median age was 50 years (range, 28–67 years) and male to female ratio was 1.5. Sokal score at the diagnosis was low in 13 patients and high in 2 patients. Four patients had been treated before IM. Previous therapies comprised IFN in all these patients, including 1 patient who relapsed after allogeneic stem cell transplantation (SCT). The clinical stage of this patient was amended to be accelerated phase at the time of diagnosis. Two other patients withdrew the consent. Excluding these 3 patients, the remaining 12 patients with low risk of Sokal score were analyzed. The median follow-up period is 23 months (range: 6–27 months). Three patients lost MMR (1, 3, and 6 months, respectively) and other 9 maintained CMR. Molecular relapse-free survival is 74%. The sustained CMR patients had the significantly longer CMR period on IM (median 31 months, range 26–79 months) compared with relapsed patients (0, 9, and 14 months, respectively; p=0.0142). There was no difference between the relapsed and the sustained CMR patients in the duration of IM treatment. All the relapsed patients achieved MMR after 2, 4, and 5 months of IM resumption, respectively. In conclusion, IFN monotherapy is a promising option for sustained molecular response after IM discontinuation in CML patients with CMR. Disclosures: No relevant conflicts of interest to declare.

Description: Previous studies have revealed various extrinsic stimuli and factors involved in the regulation of hematopoiesis. Among these, Notch-mediated signaling has been suggested to be critically involved in this process. Herein, we show that conditional inactivation of ADAM10, a membrane-bound protease with a crucial role in Notch signaling (S2 cleavage), results in myeloproliferative disorder (MPD) highlighted by severe splenomegaly and increased populations of myeloid cells and hematopoietic stem cells. Reciprocal transfer of bone marrow cells between wild-type and ADAM10 mutant mice revealed that ADAM10 activity in both hematopoietic and nonhematopoietic cells is involved in the development of MPD. Notably, we found that MPD caused by lack of ADAM10 in nonhematopoietic cells was mediated by G-CSF, whereas MPD caused by ADAM10-deficient hematopoietic cells was not. Taken together, the present findings reveal previously undescribed nonredundant roles of cell-autonomous and non–cell-autonomous ADAM10 activity in the maintenance of hematopoiesis.

Description: Abstract 3977 Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, targeting p53-independent signaling pathways is of considerable interest. We previously reported the cytotoxic efficacy of a newly discovered tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b ]dipyran-8-one (C23H26O5) (synthesized at Nippon Shinyaku, Kyoto, Japan), originally derived from Calophyllum brasiliense, with more pronounced apoptotic effects in mutant-p53 MCL cells than in wild type-p53 cells (Jin et al., ASH abstract 2009). Some of the coumarin antibiotics are known to bind to chaperone molecule 90-kDa heat shock proteins (Hsp90) and induce degradation of Hsp90 client proteins including key components of multiple signaling pathways for cell proliferation and/or survival. In this study, the mechanisms of action of GUT-70 were investigated in MCL cell lines with known p53 mutation status (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). GUT-70 demonstrated a dose-dependent binding affinity to Hsp90 (competitive binding assay using fluorescently labeled geldanamycin), increased ubiquitinated proteins accumulation, and further induced degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53, or increased Hsp70, a marker of Hsp90 inhibition. The downregulation of constitutively overexpressed cyclin D1 in MCL by GUT-70 was accompanied by p27 accumulation, decreased Rb phosphorylation, and impeded cell cycle progression in the wt-p53 JVM2 and Granta 519. However, GUT-70 induced apoptosis in mt-p53-bearing MINO and Jeko cells which was accompanied by only minimal cell cycle arrest. These findings suggest that apoptosis induction by GUT-70 in mt-p53 cells is in part independent from cell cycle arrest is known to protect cells from apoptosis. Moreover, the mt-p53 depletion by GUT-70 will further diminish its “gain of functions” for cell proliferation and anti-apoptosis. To determine if GUT-70 might potentiate the apoptotic effects of commonly used chemotherapeutic agents, we assessed the combination effects of GUT-70 with bortezomib (BTZ), a selective inhibitor of the 26S proteasome, and with doxoubicin (DOX), a conventional chemotherapeutic agent drug for MCL. Synergistic anti-proliferative effects of GUT-70 and BTZ or GUT-70 and DOX combinations were observed in both wt-p53 and mt-p53 MCL cells at 48 h post-exposure (combination index; GUT-70/Bortezomib; 0.59 for JVM2, 0.73 forMINO, GUT-70/Doxorubicin; 0.37 for JVM2, 0.35 for MINO). In conclusion, our results demonstrate that the novel anticancer agent tricyclic coumarin GUT-70, an Hsp90 inhibitor, has potential utility for mt-p53 bearing MCL cells, and in the combination therapy of MCL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5088 Aim: With the exception of CD5, the clinical implications of aberrant T-cell antigen expression on B cells in diffuse large B cell lymphoma (DLBCL) have not been well studied. The present retrospective study aimed to determine associations between T cell antigen expression and patients′ characteristics and prognosis Patients, Material and Methods: 249 cases with B-cell lymphoma (BCL) newly-diagnosed in our Institute between January 2002 and August 2009 were biopsied and also analyzed by flow cytometry. Biopsy specimens were fixed in formalin, stained with Hematoxylin-Eosin, and also immunostained. Histological subtypes were defined according to the World Health Organization Classification Version 3. Flow cytometric (F/C) analysis was performed following standard methods. The aberrant expression of one or more T cell surface antigens on B cells (CD2, 3, 4, 7, 8) was assessed. Statistical analysis was conducted to seek associations between aberrant positive and negative cases and patients′ background laboratory data, and prognosis. This study was approved by the Ethics Committee of Kitasato University School of Medicine. Results: 150 DLBCL, 68 Follicular lymphoma (FL), 17 Marginal zone lymphoma (MZL), 6 MCL and 4 CLL patients were tested. Of the DLBCL cases, 12 (8%) showed aberrant T cell antigen expression with 4, 1, 5, 1 and 1 patients′ B cells positive for CD2, CD4, CD7, CD8 and CD7 + 8 respectively. Aberrant expression among the FL, MCL, MZL and CLL cases was seen in 0, 1, 1 and 1 patient, respectively. Among the DLBCL patients, T antigen-negative cases tended to have higher WBC counts and greater CD20 expression by F/C analysis. No statistically significant associations with gender, age, IPI, clinical stage, laboratory data, expression of CD5 and other markers, ABC/GCB, or karyotype were observed between the positive and negative groups. 96 of the 150 DLBCL patients received rituximab-chemotherapy. Again, there were no statistically significant differences between the two groups in overall survival and response to treatment. Discussion: 1) CD2 and CD7 are relatively common among aberrant T cell antigen- positive DLBCL. 2) No statistically significant differences were observed between the two groups in terms of background and prognosis. 3) Both CD8-positive DLBCL patients experienced a very aggressive clinical course and rapidly succumbed to their disease. CD8-positive DLBCL cases may in general have poorer survival. 4) Further analysis will be necessary to confirm these results. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4460 The clinical use of imatinib, a specific BCR-ABL tyrosine kinase inhibitor (TKI) is effective in inducing a complete hematological and cytogenetic remission in a high percentage of chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) patients. However, imatinib does not efficiently kill leukemic stem cells and is limited by the emergence of resistance due to the point mutations in the BCR-ABL kinase domain. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. HDAC inhibitor is a structurally diverse class of targeted anti-cancer agent. One of the pan-HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid: SAHA) is a small-molecule inhibitor of most human class I and class II HDAC, and is reported the efficacy of malignant cells including lymphomas and myeloid malignancies.Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitor and an HDAC inhibitor, vorinostat may help prevent CML relapse due to BCR-ABL point mutation and may improve their long-term outcome. In this study, we investigated the efficacy of vorinostat by using the Ph-positive leukemia cell line, K562 and Ba/F3 BCR-ABL cell in a random mutagenesis study for BCR-ABL mutation. We first performed a comprehensive drug combination experiment using vorinostat and BCR-ABL tyrosine kinase inhibitor, imatinib or nilotinib. The treatment of imatinib or nilotinib exhibits cell growth inhibition partially against Ba/F3 BCR-ABL cell in a random mutagenesis. We also found the BCR-ABL point mutation such as T315I or M344V after 2 weeks nilotinib treatment by direct sequence analysis. We show that vorinostat potently induced cell growth inhibition of K562 and Ba/F3 BCR-ABL cells in a random mutagenesis in a dose dependent manner. Combined treatment of Ba/F3 BCR-ABL cell in a random mutagenesis with vorinostat and nilotinib or imatinib caused significantly more cytotoxicity than each drug alone by colony assay. We investigated the intracellular signaling of vorinostat. Phosphorylation of BCR-ABL, Crk-L were reduced after vorinostat treatment for 24 hours in a dose dependent manner. Caspase 3 and poly (ADP-ribose) polymerase (PARP) activation were increased after vorinostat treatment. Vorinostat potently enhanced cell growth inhibition of Ba/F3 BCR-ABL point mutants (G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P) compared with Ba/F3 expressing Wt BCR-ABL cells. The protein level of BCR-ABL was reduced after vorinostat treatment. BCR-ABL degradations in BCR-ABL mutant cells were significantly enhanced compared with Ba/F3 Wt BCR-ABL cells. Although long term culture of Ba/F3 BCR-ABL cell in a random mutagenesis with 2μ M vorinostat significantly decreased cell growth, the cells were increased after removal of vorinostat. We found these cells were wild type BCR-ABL by direct sequence analysis. Data from this study suggested that administration of the vorinostat may mediate its effects on BCR-ABL positive cells included BCR-ABL point mutation and enhance cytotoxic effects of nilotinib or imatinib in BCR-ABL mutant cells, and provide information of potential therapeutic relevance. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.