ALBERT

Description: Abstract 1661 Most cancer drug developments are focusing either in vitro target-based screening or cell-based phenotypic screening to identify potential compounds. The target-based screenings are powerful screening methods if the cancer relay on a single driver mutation. On the other hand, cell-based phenotypic screenings measure cell phenotypes such as growth inhibitory effect on established cancer cell lines. However, these established cell lines do not recapitulate human cancer in some aspects. For instance, most cell lines have the microenvironment-independent growth ability, while primary cancer cells can not survive in ex vivo culture. In this study, we established a new drug screening system targeting microenvironment-dependent primary lymphoma cells. First of all, we established primary lymphoma xenograft models, including three diffuse large B cell lymphoma (DLBCL), one follicular lymphoma, one intravascular large cell lymphoma (IVL), and one mantle cell lymphoma. Interestingly, lymphoma cells from the patient with severe extranodal invasion invaded extranodal organ also in mice. And pattern of invasion in tissue such as intravascular tumor invasion of IVL was maintained in the xenograft models. In addition, mRNA expression profiles were similar between primary lymphoma cells and the lymphoma cells obtained from NOG mice. Furthermore, the heterogeneity of primary tumor such as heterogenous expression of CD20 was maintained in the xenograft models. These data suggested that the lymphoma cells propagated in NOG mice kept the original patient's phenotype. Next, we investigated the microenvironment of lymphoma in primary lymphoma xenograft models. Fibroblastic reticular cell (FRC) and follicular dendritic cell (FDC) are reported to be important to support the survival of lymphoma cells in their microenvironment. In the spleen of DLBCL model mice, lymphoma cells colocalized with FRC, but FDC was not detected in NOG mouse. FRC is known to produce reticular fiber and forms fibroblastic reticular network (FRN). Lymphoma induced reticular fiber production and tumor formation on FRN were also observed in our system. Furthermore, co-culture with FRC cell line, BLS4, significantly enhanced viability of 2 out of 3 DLBCL cells obtained from primary lymphoma xenograft mice and enabled more than three weeks long-term ex vivo culture of them. These results indicated that FRC played an important role for lymphoma cell survival. Finally, we established a new drug screening system using this co-culture system. Primary DLBCL cells transplanted to NOG mice were collected and seeded on pre-seeded BLS4 in 96-well plates (lymphoma cell co-culture). Monoculture of BLS4 in 96-well plates was also prepared. Both lymphoma co-cultured cell and BLS4 monoculture cell were treated by 2613 kinds of pharmacologically active compounds for 72 h. DAPI-stained dead cells of lymphoma cells were counted by image analyzer, and the proliferation of BLS4 was measured by MTT assay. We calculated Drug Effect Index (DEI) by multiplication of dead cell number of lymphoma cells and MTT value of BLS4 and compared. The compound with the highest DEI was pyrvinium pamoate, oxyuricide. Pyrvinium pamoate aborogated the survival of lymphoma cells co-cultured with BLS4 dose-dependently. And subcutaneous tumor of primary lymphoma cells and BLS4 were diminished by single intratumoral injection of 20 mg/kg pyrvinium pamoate. These results indicated that our screening system could be performed in large scale and select drugs with anti-tumor activity to primary lymphoma cells. Screening against primary lymphoma cells sheds new light on lymphoma drug development. Disclosures: Sugimoto: Otsuka Pharmaceutical co ltd: Employment.

Description: Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

Description: Abstract 3227 PAX5 is a transcription factor expressed in B lymphoid lineage from pro-B cell to mature B cell, and is required for B-cell development and maintenance. De-regulated and reduced PAX5 activity has been implicated in B cell malignancies both in human disease and mouse models. Furthermore, the PAX5 gene is the most frequent target of somatic mutations in childhood and adult B-progenitor acute lymphoblastic leukemia (ALL), being altered in 38.9% and 34% of cases, respectively. These mutations consist of partial or complete hemizygous deletions, homozygous deletions, partial or complete amplifications, point mutations or fusion genes. These aberrations of the PAX5 gene will impair PAX5 function more or less, and will be involved in the block of B-cell differentiation. Chromosomal translocation t(9;15)(p13;q24) was found in 2 cases of childhood ALL and resulted in an in-frame fusion of PAX5 to PML gene. PAX5 moiety of PAX5-PML retains its DNA binding domain but loses its transactivation domain suggesting PAX5-PML will be a dominant negative form of PAX5. PML is originally found as a fusion partner of RARα in PML-RARα, an oncoprotein found in acute promyeloid leukemia (APL), and is now thought to be a tumor suppressor and a pro-apoptotic factor. PML-RARα dominant-negatively affects PML function by disrupting PML nuclear bodies (NBs) where PML exerts its function and gives APL cells survival advantage. These findings give rise a speculation that PAX5-PML not only causes differentiation block by transcriptional repression of PAX5 target genes but also confers a survival advantage by inhibition of PML function. However, no functional analysis has been done for PAX5-PML. Here, we demonstrate that PAX5-PML had a dominant negative effect on both PAX5 and PML. PAX5-PML inhibited transcriptional activity of PAX5 in luciferase reporter assay. PAX5-PML expression also suppressed expression of CD19, one of the transcriptional targets of PAX5, in B-lymphoid cell line. Surprisingly, PAX5-PML hardly showed DNA binding activity in electro mobility shift assay although it retains DNA binding domain of PAX5. Further detailed analyses including, luciferase assay using PAX5-PML with DNA-binding-dead mutations, co-IP assay, and ChIP assay, suggested that transcriptional repression by PAX5-PML was independent of its DNA binding ability, that PAX5 and PAX5-PML formed a heterodimer, and that PAX5-PML bound to CD19 promoter through the association with PAX5 on the promoter. On the other hand, co-expression of PAX5-PML inhibited PML sumoylation, an essential post-translational modification for PML to form NBs, and altered PML localization from NB pattern to diffuse nuclear pattern. Furthermore, treatment with arsenic trioxide, a therapeutic reagent for APL that induces enhancement of PML sumoylation, reconstitution of PML NBs, and apoptosis in APL cells, induced recovery of PML sumoylation and reconstitution of PML NBs also in cells expressing PAX5-PML. More importantly, PAX5-PML expressing HeLa cells showed resistance to PML dependent apoptosis such as apoptosis induced by irradiation and histone deacetylace inhibitor. And arsenic trioxide treatment overcame these apoptosis resistance conferred by PAX5-PML. These data suggest the involvement of this fusion protein in the leukemogenesis of B-ALL in dual-dominant negative manner and the possibility that some cases of ALL can be treated with arsenic trioxide. Disclosures: Hayakawa: Otsuka: Research Funding. Naoe:Chugai: Research Funding; Zenyaku: Research Funding; Kyowa-Kirin: Research Funding; Dainippon-Sumitomo: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Otsuka: Research Funding.

Description: Introduction CD7 is expressed on human T and NK cells and progenitors of myeloid cells as well as blasts in some patients with myelodysplastic syndromes (MDS). We showed previously that CD7 positivity of MDS blasts was associated with aggressive characteristics and poor prognosis. However, the mechanisms by which CD7-expressing MDS blasts are associated with disease progression remain unknown. In this study, we first investigated the biology of CD7-expressing MDS blasts. Second, to validate the prognostic impact of CD7 on MDS blasts, prognostic variables including the CD7 positivity of MDS blasts and a new prognostic system, the revised International Prognostic Scoring System (IPSS-R) were evaluated in Japanese MDS patients. Methods & Results 1) To investigate the mechanisms regulating CD7 expression, we used MDS cell lines HNT-34 and F-36P. CD7 expression on these cells was partially down-regulated by inhibition of NFκB. 2) To investigate the survival potential in CD7+ MDS blasts, we analyzed cell cycles and spontaneous apoptosis by flow cytometry. CD7+ blasts had a cell cycle advantage compared with CD7– blasts in F-36P but not in HTN-34 cells. Compared with CD7– HNT-34 cells, CD7+ HTN-34 cells were more resistant to spontaneous and serum deprivation-induced apoptosis. 3) We then compared gene and protein expression levels of apoptosis-related proteins including Bad, Bax, Bcl-2-L1, Bcl-2, caspase-3, caspase-8, caspase-9, FADD, Fas, and FasL between CD7+ and CD7– blasts of these cell lines using real-time PCR and flow cytometry, respectively. CD7– blasts had markedly higher expression levels of the Bad gene and protein compared with CD7+ blasts in both cell lines. The expression levels of Fas and FasL were suppressed in CD7+ blasts compared with CD7– blasts in F36P cells. These results support the association of CD7 expression on MDS blasts with disease progression. 4) To reevaluate the prognostic impact of CD7 expression as well as R-IPSS, prognostic variables were analyzed in 81 MDS patients [refractory anemia (RA) 55, RA with ringed sideroblasts 7, RA with excess blasts (RAEB) 18, and RAEB in transformation 1], comprising 50 men/31 women with a median age of 67 (range 27–88) years. Immunophenotyping was performed by 3-color flow cytometry, in which blast cells were gated with a CD45-gating method, and 9 parameters including CD7 expression on MDS blasts, i.e., CD34+ myeloblast-related and B-progenitor-related cluster size, myeloblast CD45 expression, and aberrant expression of CD7, CD10, CD11, CD15, CD56, and B7-H1 on myeloblasts, were analyzed. Using the Cox proportional hazard regression model, we identified five prognostic variables: IPSS-R score; percentage of blasts in peripheral blood; CD7 expression; gender; and white blood cell count. The chi-square test showed that the IPSS-R score and CD7 expression were strong prognostic factors (P = 0.0114 and 0.006, respectively). Patients whose MDS blasts expressed high levels of CD7 (17% or more MDS blasts were CD7+) had significantly shorter survival than other patients. Conclusion Our study revealed that CD7+ MDS blasts had apoptosis resistance with decreased expression of apoptosis-related genes, especially Bad. Signaling via CD7 on MDS blasts might inhibit Bad expression and then confer apoptosis resistance. Further studies are in progress to clarify CD7 signaling in cell lines as well as in MDS blasts from patients. Furthermore, we demonstrated for the first time that IPSS-R as well as CD7 expression on blasts had a strong impact on MDS patient prognosis. Disclosures: Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding. Shibayama:celgene: Honoraria, Research Funding; Janssen: Honoraria. Naoe:Otsuka Pharmaceutical Co., Ltd, Kyowa Hakko Kirin Co., Ltd., Wyeth, and Chugai Pharmaceutical Co., Ltd.: Research Funding.

Description: Abstract 3527 With modern intensive chemotherapy, about 90% of adult patients with Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) achieve complete remission. However, the overall survival rate drops due to the high rate of relapse. Therefore, the establishment of optimal post-remission therapy is important, and the efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-matched sibling in first remission (CR1) has been demonstrated through clinical studies using genetic randomization. However, the efficacy of unrelated HSCT for adult patients with Ph-negative ALL in CR1 who lack an HLA-matched sibling remains unclear. Decision analysis is a statistical technique that aids the clinical decision-making process under uncertainty, especially in situations where a well-designed clinical trial is practically difficult to perform. We previously demonstrated through a decision analysis that HSCT is superior to chemotherapy (CTx) alone in CR1 for adult patients with Ph-negative ALL who have an HLA-matched sibling, even after an adjustment for quality of life (QOL) (Kako S et al, BMT 45 supp. p414, 2010). In a similar manner, we performed a decision analysis based on the decision tree (Figure 1) to evaluate the efficacy of unrelated HSCT for adult patients with Ph-negative ALL in CR1 who lack an HLA-matched sibling. The transition probabilities and utilities were estimated from studies of the Japan Adult Leukemia Study Group (JALSG) (ALL93; n=122, ALL97; n=119), the database of the Japan Marrow Donor Program (JMDP) (HSCT in CR1: n=231), and the literature. The primary outcome measure was the 10-year survival probability with or without a QOL adjustment, in which we especially consider the presence of chronic graft-versus-host disease (GVHD). Subgroup analyses were performed according to risk stratification based on the white blood cell count and cytogenetics, and according to age stratification with a cutoff of 35 years. In the whole population, the superiority of unrelated HSCT in CR1 was demonstrated in analyses both with and without a QOL adjustment (40.6% vs. 31.1% and 43.6% vs. 31.9%, respectively). A probabilistic sensitivity analysis using a Monte Carlo simulation supported these results. A similar tendency was observed in all subgroups (Table 1). The decision model was sensitive to the probability of disease-free survival following CTx and the probability of overall survival following HSCT in CR1 in standard-risk and higher-aged patients. In conclusion, to improve the probability of long-term survival, unrelated HSCT in CR1 is recommended for patients who lack an HLA-matched sibling donor. However, improvement of CTx in the future may change the result. Table 1. Expected 10-year survival probabilities with and without adjusting for quality of life (QOL) Expected survival probability without a QOL adjustment Expected survival probability With a QOL adjustment HSCT Chemotherapy HSCT Chemotherapy All patients 43.6% 31.8% 40.6% 31.0% Standard-risk patients 44.0% 36.7% 40.9% 36.0% High-risk patients 43.7% 23.8% 40.6% 23.1% Lower-aged patients 44.3% 30.0% 41.2% 29.2% Higher-aged patients 42.1% 33.1% 39.1% 32.6% Abbreviations: HSCT, hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3898 Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of Blimp1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of Blimp1 and XBP-1 and subsequent suppression of Pax5 by Blimp1 were observed and thought to be the trigger of plasma cell differentiation. However, the first trigger that represses PAX5 function is yet to be revealed. Here, we investigated the relevance between Pax5 and MAP kinase signal, a main component of BCR signal. We demonstrated PAX5 phosphorylation by ERK2 in vitro and in vivo (Figure. A). The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In the luciferase reporter assays, PAX5 phosphorylation by MAP kinase signal reduced the transcriptional activity of PAX5 when CD19 promoter sequence was used for reporter gene. On the other hand, PAX5 caused transcriptional repression of Blimp1 promoter, which was released by PAX5 phosphorylation by MAP kinase signal. These data supported the reported findings that PAX5 had dual function, and suggested that the PAX5 function was inhibited by its phosphorylation. We demonstrated that B cell receptor stimulation with anti-IgM antibody induced strong MAP kinase activation, phosphorylation of endogenous Pax5 (Figure. B), and upregulation of Blimp1 mRNA (Figure. C). All of these phenomena were inhibited by U0126, MEK inhibitor. These results suggested that transcriptional repression of Blimp1 by PAX5 was released by its phosphorylation by MAP kinase signal. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells. Disclosures: Hayakawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

Description: Abstract 2874 Background: Rituximab is an anti-CD20 mouse/human chimeric monoclonal antibody. Recent reports indicate that combination chemotherapy with rituximab for CD20(+) B-cell lymphoma has improved the clinical outcomes compared to conventional chemotherapy without rituximab. However, patients showing rituximab resistance have been also recognized. We previously reported observation of relapse (RD)/progression (PD) with CD20(-) phenotype after using rituximab, and CD20(-) phenotype is closely related to ritxuximab resistance (Hiraga J, et al., Blood, 2009., Sugimoto T, et al., Biochem Biophys Res Commun, 2009., Tomita A, et al., Int J Hematol, 2007.). We concluded that aberrant down-regulation of MS4A1 (CD20) gene expression was one of the critical mechanisms of CD20(-) phenotypic change. Moreover, all those patients died of disease progression within one year after CD20(-) change, indicating the tendency for a relatively poor prognosis. Since such patients occur less frequently, more patients are definitely needed to confirm the clinical features and molecular basis. Aims: To confirm the clinical features of patients who show CD20-protein-negative phenotypic change after using rituximab. Analyses of the molecular backgrounds of CD20(-) phenotypes will be conducted using CD20(-)-transformed primary lymphoma cells. Results: CD20(+) B-cell lymphoma patients (DLBCL, FL, MALT, MCL, CLL/SLL, and Burkitt) treated with rituximab containing chemotherapy, were used for the analyses. CD20 protein expression was confirmed by immunohistchemistry (IHC) and flow cytometry (FCM) using primary lymphoma cells at the time points of the initial diagnosis and the RD/PD. All patients analyzed showed CD20 IHC(+)/FCM(+) phenotype at the initial diagnosis, and the CD20(-) change was confirmed by IHC(-)/FCM(-) at RD/PD period. Ten patients showed CD20(-) phenotypic change after using rituximab. Pathological diagnosis of those patients was DLBCL (8 out of 10 cases; 8/10) and FL (2/10) at the initial diagnosis, and DLBCL (10/10) at CD20(-) change in RD/PD period. TdT was negative by IHC in 6 out of 6 analyzed cases. Bone marrow (BM) invasion at diagnosis was confirmed in 9/10, and extranodal involvement (BM, CNS, skin, and peripheral blood) at CD20(-) RD/PD period was confirmed in all patients (10/10). Leukemic transformation at the terminal stage was seen in 6/10 patients, and LDH elevation seems to reflect their disease status. All 10 patients died of disease progression within 1 year after the diagnosis of CD20(-) phenotypic change in spite of salvage chemotherapy. Molecular backgrounds were analyzed in patients whose primary lymphoma cells were available for molecular analyses. Quantitative RT-PCR indicated that the CD20 mRNA expression level was significantly lower than that of CD20 positive controls in all analyzed cases (5/5). Almost the same result was obtained by immunoblot analysis using anti-CD20 C-terminus antibody. Genetic mutations on the coding sequence (CDS) of MS4A1 gene were suggested in 2 out of 7 cases, but the frequency of the mutated clone was less than 20%. CD79b ITAM mutation was confirmed in 1 out of 5 cases. In vitro rituximab binding assay using fluorescent-labeled-rituximab and primary lymphoma cells indicated that the rituximab binding was significantly lower on CD20(-) cells compared to the control cells. Conclusions and Discussion: All the patients showing CD20(-) phenotypic change were diagnosed as DLBCL, and extranodal infiltration was confirmed in all cases. Leukemic transformation was relatively common at the terminal stage, and LDH elevation seemed to reflect the disease progression. CD20 mRNA expression level was generally lower than that of positive control cells, and clinically significant MS4A1 gene mutations were not confirmed. All the patients died within 1 year after diagnosis of CD20(-) transformation, suggesting that the CD20(-) change may closely correlate with the poor prognosis. From these findings, the phenotypic change after using rituximab may be considered as a particular clinicopathologic group such as “post-rituximab extranodal CD20-negative lymphoma.” Further accumulation of patients is warranted. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Description: Abstract 3417 Background: Now, imatinib has been established as a first line therapy for the patients with chronic myelogenous leukemia (CML) in chronic phase (CP), and a standard initial dose is 400 mg daily. Although less than 300 mg showed unsatisfactory results, 300 mg or more appears effective in some patients intolerant to standard dose. However, large studies have not yet explored the response to lower dose of imatinib. In this CML202 study, although initial dose of imatinib was scheduled to be 400 mg, many patients actually received reduced dose mainly due to adverse events. However, overall efficacy and outcomes had been comparable to other studies. We performed subgroup analysis regarding long-term survivals according to the mean daily dose during the first 6 months, 12 months, and 24 months, respectively. We also measured imatinib plasma trough concentration (Cmin) in patients receiving imatinib at a dose of 300 mg or 400 mg, and compared efficacy and survivals between them. Methods: The prospective multicenter study of imatinib therapy in Japanese patients with newly diagnosed CML-CP was conducted in Japan Adult Leukemia Study Group (JALSG CML202 study). The objectives of this study were to determine the efficacy, safety and long-term outcomes of imatinib therapy in patients with newly diagnosed CML-CP. Primary end point of imatinib therapy was overall survival (OS). Initial daily dose of imatinib was 400 mg. The plasma concentration of imatinib was measured using liquid chromatography-tandem mass spectrometry. Results: 488 patients were enrolled between 2002 and 2006, and data at a median follow-up of 66 months were analyzed. The cumulative best response rates of major cytogenetic response (MCyR), complete cytogenetic response (CCyR) and major molecular response (MMR) were 99%, 98% and 81%, respectively. At 66 months, the estimated rate of overall survival (OS) was 93% (95% CI, 90 to 96), and that of survival without progression to accelerated phase (AP) or blast crisis (BC) was 97% (95% CI, 96 to 99). In the landmark analysis at 12 months or 18 months according to the cytogenetic response or the molecular response, patients who had CCyR or MMR showed significantly better event free survival (EFS) rates than those who had not, respectively. Mean daily doses of imatinib administered during the first 24 months were 400 mg or more (400 mg group) in 294 patients, less than 400mg and 300mg or more (300 mg group) in 108 patients, and less than 300 mg (200 mg group) in 86 patients. The efficacy and outcomes at 66 months were evaluated according to the mean daily dose (400, 300, 200 mg group). The estimated rates of OS were 98%, 92% (P=0.09), and 74% (P

Description: Abstract 4457 Chronic myeloid leukemia (CML) is effectively treated with imatinib (IM), however, several mathematical models and ex vivo-examinations suggested that IM-therapy does not eradicate BCR-ABL-positive hematopoietic stem cells (HSC). We prospectively (0, 3, 6 and 12 months after IM-therapy) investigated 16 newly diagnosed and 22 long-term followed CML-chronic phase (CP) cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) (Figure 1) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM-therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations while gradual reduction was observed during the first 12 months in all populations. And discrepancy of minimum residual diseases (MRD) between the HSC populations and other populations was larger in patients after longer IM-therapy. In evaluating properties of CML stem cells and other markers, we observed irrelevant distribution of side population (SP) and expressions of ABC transporters (ABCB1 and ABCG2) in comparison with CD34/38 expression. We also prospectively investigated BCR-ABL transcripts in each population of 23 IM-resistant or -intolerant CML-CP cases and one newly diagnosed CML-accelerated phase (AP) case during treatment with second-generation tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months and there was no significant difference of MRD among each population in optimal responders to 2nd TKIs-therapy. In the stromal co-culturing system using primary cells and leukemic NOD/SCID/IL2rgnull (NOG) mice xenotransplanted with Ph+ leukemia cells, retention of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ population after IM-treatment were observed and cell death mechanisms after treatment with 2nd TKIs are also under investigation. These results imply that therapy with 2nd TKIs could be a promising approach for quick and efficient reduction of the CML stem cells and cure of disease. Figure 1 Figure 1. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Description: Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.