ALBERT

Description: Natural killer (NK) cells are components of the innate immunity and play an important role in cancer surveillance through their cytolytic and immunomodulatory capabilities. Infusion of NK cells is a promising tool for cell therapy of hematologic malignancies and solid tumors. However, the potent cytotoxicity of NK cells might be hampered by tumor immune escape mechanisms and intrinsic resistance. We and others previously demonstrated intrinsic resistance of leukemia cells to NK cell lysis can be overcome by the transduction of artificial antigen receptor into NK cells. The genetic engineering of primary NK cells with chimeric antigen receptor improved cytotoxic activity and cytokine production, and this enhanced function was target-specific. Thus, a novel method to enhance NK cell activity against a wide range of tumors is also required. Several cytokines are associated with enhanced cytotoxicity, in vivo survival, and proliferation of NK cells. In particular, interleukin (IL)-21, which shares the common cytokine-receptor gamma chain with IL-2, was reported to enhance the cytotoxicity of human NK cells. In the present study, we investigated whether the enforced expression of human IL-21 in primary human NK cells enhanced their cytotoxicity against leukemic cells and allowed prolonged survival. We collected peripheral blood samples from healthy adult donors, and mononuclear cells were isolated by density gradient centrifugation. Primary NK cells were expanded by stimulation with K562-mbIL15-41BBL cell line following standard procedures. After 7 days of expansion, residual T cells were removed with magnetic beads and NK cells were transduced with a retroviral vector containing human IL-21 cDNA and GFP. Fourteen days after transduction, more than 95% of cells were CD56+CD3- NK cells. Median GFP expression in the CD56+CD3- cells was 84.2% (74.5%-97.1%, n=6). We confirmed that NK cells transduced with human IL-21 cDNA (NK-IL21) had intracellular expression of IL-21 as assessed by flow cytometry, while NK cells transduced with a vector containing GFP only (NK-mock) did not. 4-hour cytotoxicity assays revealed significantly enhanced cytotoxicity exerted by NK-IL21 (Fig. 1). Cytotoxic activity of NK-IL21 against K562 cells and Jurkat cells was significantly higher than that of NK-mock. We found that the intracellular expression levels of both perforin and granzyme B were higher in NK-IL21 cells than in NK-mock cells, in accordance with their higher cytotoxicity against target cells. However, NK-IL21 did not show increased expression of the apoptosis-inducing molecule TRAIL, NK cell activating receptor NKG2D, or natural cytotoxicity receptors p30, p44, or p46. The success of NK cell infusions might rely on the in vivo persistence of NK cells. We therefore tested whether the enforced expression of IL-21 in NK cells enhanced their proliferation and survival, and found that IL-21 expression in NK cells did not prevent apoptosis induced by IL-2 withdrawal and therefore did not favorably alter cell proliferation without IL-2. In contrast to the favorable results obtained by short-time cytotoxicity assays, NK-IL21 did not exert effective tumor control in long-term coculture experiments. The residual leukemic cell burden in NK-IL21 cocultures was not decreased and did not differ from that in NK-mock coculture experiments where cocultures were extended to 7 days without IL-2. However, by adding IL-2 (100 U/ml) to the culture, we demonstrated a dramatic suppression of residual leukemia burden exerted by NK-IL21. As shown in Figure 2, the number of residual K562 cells in the NK-IL21 cocultures was much lower than in the NK-mock cocultures (1.9% ± 0.4% vs 61.5% ± 3.8% of control culture without NK cells at a 1:1 E:T ratio, p

Description: BACKGROUND: The trial JACLS ALL-02 for treatment of childhood acute lymphoblastic leukemia (ALL) was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in unfavorable-risk groups by treatment intensification. These aims were pursued through a refined stratification strategy using white blood cell count, age, immunophenotype, unfavorable genetic aberrations, and treatment response providing an excellent discrimination of risk groups. PATIENTS AND METHODS: Between April 2002 and March 2008, 1252 children with newly diagnosed de novo ALL with 1-18 years of age were enrolled to JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Patients with BCP-ALL were stratified into 3 groups: standard-risk (SR), high-risk (HR), extremely high-risk (ER), based on initial prednisolone (PSL) response and the modified National Cancer Institute(NCI) workshop criteria. Prednisolone poor response (PPR) was determined after 7 days of monotherapy with prednisone and one intrathecal dose of methotrexate. PSL good responders (PGR; 〈 1,000 blasts/microL) were divided into SR and HR according to the modified NCI workshop criteria by WBC 10K and age 10, and received conventional therapy. BCP-ALL with PPR (≥ 1,000 blasts/microL) or t(4;11), and acute mixed lineage leukemia/ acute unclassified leukemia were assigned to ER and received intensified post-induction therapy . Patients with T-ALL were treated by a specific protocol that was different from the protocol used for BCP-ALL. Bone marrow response was also evaluated in aspiration smears on day 15 and 33 of induction treatment, and those who had slow early bone marrow response, defined as an M3 marrow on day 15 or M2/3 marrow on day33, shifted to the higher risk and received augmented post-induction therapy. Cranial irradiation was restricted to patients with initial central nerve system involvement or T-ALL with high WBC (≥10K) at diagnosis. Alternatively, protracted TIT was given during induction, intensification and maintenance depending on the risk group (12 doses in SR/T and 15 in HR/ER). Slow early responders who had an M3 marrow on day 15 in ER/T or M2/3 marrow on day33 in any risk, underwent to stem cell transplantation (SCT). PPRs without slow early bone marrow response underwent to SCT only if a matched sibling donor was available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTS: Estimated 4-year EFS and OS for all 1252 patients was 83.7% (SE=1.1) and 90.3% (SE=0.89), slightly better in EFS than the former study (ALL-97; 79.3%, SE=1.7, p=0.054). The SR group (N=457, 37%) achieved excellent 4-year EFS of 90.4% (95%CI: 87.2, 92.7) and 4-year OS 97.3% (95.3, 98.5); in SR without slow early bone marrow response, patients with hyperdiploid and triple trisomy showed excellent 4-year EFS/OS of 100%. In the HR group (N=542, 43%), the estimated 4-year EFS and 4-year OS were 84.9% (81.5, 87.7) and 89.3% (86.3, 91.6), respectively; older age (≥ 6 years) was predictive for inferior OS (hazard ratio=1.14, p

Description: Introduction Clinical outcome of relapsed pediatric B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remains poor, although survival rate for children with BCP-ALL has greatly increased over time and is now reached 90%. To clarify the molecular pathogenesis of relapsed ALL may provide novel prognostic markers and therapeutic targets. Some genome-wide analyses for specific patients group with poor prognosis, such as early-relapsed patients and Ph- or BCR-ABL-like patients, were reported. They described important insights to understand genetic background of poor prognosis. However, the majority of relapsed cases did not have any poor prognostic marker, and the molecular mechanisms of relapse in these cases still remained unclear. Therefore we performed whole exome sequencing (WES) to describe clonal evolution in 21 relapsed pediatric BCP-ALL patients. Our cohort included various cases whose time to relapse from diagnosis were between 6 months to over 10 years. We also analyzed the clonality of leukemia cells using immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements. Patients and Methods Genomic DNA was isolated from 21 cases whose median time to relapse was 33 months. Somatic mutations including SNVs (single nucleotide variants), insertions / deletions and CNVs (copy number variants) were detected by WES using Agilent SureSelect and illumine HiSeq systems. To evaluate accurate VAF (valiant allele frequency), targeted deep sequencing was performed in candidate somatic mutations. The clonality analysis of leukemia cells was performed by standard PCR methods using Ig and TCR rearrangements. Results WES was performed in samples obtained at diagnosis, remission and relapse from 21 pediatric BCP-ALL patients. Tumor specific mutations had been identified by WES. Three of 21 were hypermutated with over 150 somatic mutations at relapse. Mutation of DNA mismatch repair gene, MSH3, was detected in 2 of them. Except for these hypermutated cases, the median number of somatic mutations detected at relapsed phase was 22 (range 8 to 53), which was higher than that at diagnosis (median 16, range 6 to 31). Sixteen recurrently mutated genes were identified in 21 cases by WES. Some known leukemia associated genes were detected, including KRAS and WHSC1 observed only at diagnosis and IKZF1 and CREBBP observed at relapse. Then we compared VAFs of these mutations between at diagnosis and relapse to solve the clonal architectures over time. Three patterns of clonal evolution were estimated from VAFs using targeted deep sequencing; (i) In 7 cases, all mutations described at diagnosis were shared at relapse, suggesting that relapse clone derived from predominant clone at diagnosis with additional mutations in these cases. (ii) In other 13 cases, most of mutations in predominant clone at diagnosis were not detected at relapse except for some shared mutations at diagnosis and relapse, indicating that relapsed clone occurred from founder clone existing as subclone at diagnosis. (iii) In one very late-relapsed case, there were no shared mutations at diagnosis and relapse. According to clonality analysis of Ig and TCR, none of rearrangements identified at diagnosis were conserved at relapse in this case. On the other hand, most rearrangement at diagnosis were conserved at relapse in other 20 cases except one patient who relapsed in 10 years after diagnosis. Relapse from predominant clone at diagnosis were observed in only one out of 8 late-relapsed cases (〉 36 months), whereas a half of the early-relapsed showed this clonal evolution pattern. The number of shared mutations between diagnosis and relapse was very limited in very late-relapsed cases over 10 years. Discussion Our study suggests that the clonal evolution pattern differs according to the time to relapse. In a half of early-relapsed cases, relapsed clone derived from major clone at diagnosis with additional mutations, and clonal selection of resistant clones occurred during treatment. Meanwhile, in late-relapsed cases, relapse was frequently associated with clonal evolution from minor subclone with some conserved mutations and same Ig/TCR rearrangements. The founder clone should be remained dormant for a long period until additional mutations lead to relapse. Towards a better understanding of clonal evolution in ALL, our study will shed light on the early prediction of relapse risk and new treatment strategies for relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: Objective: The Myc proteins are transcription factors with essential roles in cell growth and proliferation through their ability to regulate gene expression. MYC binding protein 2(MYCBP2) is probable E3 ubiquitin-protein ligase and its function in leukemia is undetermined. IKZF1 encodes a kruppel-like zinc finger protein Ikaros that is essential for normal hematopoiesis and acts as a tumor suppressor in acute lymphoblastic leukemia(ALL). IKZF1 deletion is associated with the development of ALL and poor clinical outcome. This study aimed to explore the expression of c-MYC and MYCBP2 and their correlation with clinical features in adult ALL, as well as the mechanism by which Ikaros directly regulates c-MYC/MYCBP2 expression in ALL. Methods: Quantitative PCR (qPCR) was performed to explore the expression of c-MYC and MYCBP2 in 151 newly diagnosed adult patients with ALL. The correlations of c-MYC/MYCBP2 expression with clinical parameters and survival status were analyzed. In addition, luciferase assay, quantitative Chromatin Immunoprecipitation (qChIP) and Ikaros shRNA knockdown were performed to further explore the mechanism underlying regulation of c-MYC/MYCBP2 expression. Results: Expression of c-MYC is significantly higher and MYCBP2 is significantly lower in both B-ALL and T-ALL patients compared with that in normal controls. C-MYC expression is also negatively co-related with the MYCBP2 in ALL cohorts. The patients with c-MYC high and MYCBP2 low expression (c-MYChigh +MYCBP2low) showed higher median white blood cell counts (WBC) (101.5×109/L vs 29.4×109/L, P =0.007), incidence of splenomegaly and liver infiltration (75.0% vs 33.3%, P =0.004;75.0% vs19.4%, P =0.000), percentage of CD34(+) and CD33(+) cells (90.0% vs 61.3%, P =0.025; 80.0% vs 25.8%, P =0.000) and a lower percentage of complete remission (CR) rate (60.0% vs 92.0%,P =0.027) compared with that of patients with c-MYC low and MYCBP2 high expression (c-MYClow +MYCBP2high). Notably, our Ikaros ChIP-seq data showed strong Ikaros binding peaks in the promoter region of both c-MYC and MYCBP2. The qChIP assay showed that Ikaros significantly binds to c-MYC and MYCBP2 promoter regions in both Nalm6 B-ALL and Molt4 T-ALL cells. Moreover, expression of Ikaros suppressed c-MYC but increased MYCBP2 expression in both Nalm6 and CEM T-ALL cells. Conversely, Ikaros knockdown induced the increase of c-MYC but decrease of MYCBP2 in Nalm6 and CEM cells. Ikaros activator,Ck2 inhibitor TBB suppress c-MYC and increase MYCBP2 expression in a dose-dependent manner in Nalm6 and CEM cells. Ikaros knockdown with shRNA could block the TBB-induced suppression of c-MYC and increase of MYCBP2 expression. These data indicated that both c-MYC and MYCBP2 are direct Ikaros targets in ALL and Ikaros regulates their expression. Importantly, we also observed Ikaros binding to c-MYC and MYCBP2 promoters in primary B-All and T-ALL. The expression of c-MYC significantly increased and MYCBP2 decreased in patients with Ikaros deletion compared to that of Ikaros wild type. These data indicated Ikaros regulatory effect on c-MYC and MYCBP2 in ALL patients and Ikaros deletion is one of the reasons for expression change of c-MYC and MYCBP2 in the patients. Conclusion: We observed the expression of c-MYC significantly increased and MYCBP2 decreased in adult ALL patients. C-MYC high and MYCBP2 low expression is correlated with high-risk leukemia. Ikaros dysfunction is one of the reasons underlying c-MYC high and MYCBP2 low expression in the patients. Our data revealed the oncogenic effect of Ikaros/MYCBP2/c-MYC on oncogenesis in adult ALL, also suggested CK2 inhibitor exert its anti-leukemia effect through Ikaros-mediated regulation on c-MYC and MYCBP2 expression in leukemia. Disclosures No relevant conflicts of interest to declare.

Description: Introduction B-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner. Methods We enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection. Results The median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]). Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation. Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified. Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q 〈 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL. Conclusions We revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Description: Introduction: Genetic abnormalities are important to predict prognosis and sometimes can be therapeutic target in pediatric acute lymphoblastic leukemia (ALL). Although the cell lines with recurrent chromosomal abnormalities or leukemic fusions derived from ALL patients are useful tool for various in vitro experiments, it has not been fully investigated whether there is the difference of genetic alterations between clinical samples and cell lines.Here, we performed MLPA analysis of 86 ALL cell lines to determine copy number abnormalities (CNA) and compare with those of the patient's clinical samples. Methods: We performed MLPA analysis of 86 cell lines of ALL (14 with BCR-ABL, 11 with MLL rearrangement, 18 with TCF3-PBX1, 4 with TCF3-HLF, 4 with ETV6-RUNX1 and 35 B-other ALL cell lines) to determine CNA of IKZF1, PAX5, CDKN2A, CDKN2B, ETV6, RB1, BTG1 and EBF1. Then, CNAs were compared to those of patients' samples such as UK cohort (Schwab C, et al. Haematologica, 2013) and Japanese cohort (Asai D, et al. Cancer Med, 2013) according to each specific genetic abnormality, such as BCR-ABL, ETV6-RUNX1, TCF3-PBX1 and MLL-related fusions. In addition, we performed multiplex PCR and RNA-seq to determine fusion transcripts related to Ph-like ALL for the six Ph-negative cell lines with IKZF1 deletions. To determine the expression level of IKZF1 isoform in these cell lines, we performed real time PCR analysis of IKZF1 isoform 1 (IK1) and isoform 6 (IK6). Results: In the BCR-ABL positive cell lines, the frequencies of CDKN2A/2B and BTG1 deletion significantly higher than those in UK cohort (CDKN2A/2B: 100 vs 48%, P

Description: Introduction: Tyrosine-kinase inhibitor (TKI) treatment generally induces a good response and shows long-term efficacy in patients with chronic myeloid leukemia in chronic phase (CML-CP). The ENESTnd trial demonstrated that nilotinib was superior to imatinib in terms of inducing rapid and deep responses and preventing transformation to acute phase/blast crisis in patients with untreated CML-CP. The optimal goal of CML-CP treatment is to cure the patient, such that they remain tumor free without continuing TKI treatment. The STIM trial showed that continuous complete molecular response (CMR) is essential for discontinuing TKI, while a major molecular response (MMR) is insufficient. Aim: Here we evaluated the efficacy and safety of a change to nilotinib in Japanese patients who achieved MMR with continuous detectable BCR-ABL1 transcript levels after imatinib treatment. Patients and methods: Patients with CML-CP who had achieved MMR but not CMR (MR4.5) after over 18 months of imatinib therapy were switched to treatment with nilotinib 400 mg twice daily for up to 24 months. BCR-ABL1 transcript levels were assessed every three months. The primary endpoint was the accumulative rate of CMR (MR4.5) up to 24 months after initiating nilotinib. Results: Between Feb. 2012 and Jan 2014, 53 patients were enrolled, of whom 39 met the eligibility criteria. We evaluated a total of 38 patients with a median age of 57.5 years (range, 22-76 years). Of these 38 patients, 27 completed 24 months of nilotinib treatment. The remaining 11 discontinued nilotinib due to retraction of consent (3 patients), MMR loss (1 patient), intolerance (3 patients), or adverse events (5 patients). Twenty patients (52.6%, 90% CI: 33.3-61.8%) achieved CMR (MR4.5). The accumulative incidences of achieving CMR (MR4.5) by 3, 6, 9, 12, 15, 18, 21, and 24 months were 21.1%, 34.2%, 42.1%, 47.4%, 47.4%, 47.4%, 47.4%, and 52.6%, respectively. None of the patients transformed to acute phase/blast crisis. Neither the periods of achieving CCyR nor MMR correlated to the incidence of achieving CMR (MR4.5) after switching from imatinib to nilotinib. The adverse events were consistent with those found in other nilotinib studies: grade 3/4 adverse events included anemia (5.3%), neutropenia (2.6%), thrombocytopenia (2.6%), lipase increase (5.3%), hypoglycemia (5.3%), hypophosphatemia (39.5%), ascites (2.6%), AST, ALT increase (2.6%), pulmonary edema (2.6%), muscle pain (2.6%), nausea (2.6%), skin rash (2.6%), dyspnea (2.6%), and hyponatremia (2.6%). Cardiovascular events included atrial fibrillation (G2), chest tightness and dyspnea (G1), myocardial infarction (G2), and heart failure (G3), which occurred in one patient each and the latter three complications led to nilotinib discontinuation. Discussion and Conclusion: This NILSw trial showed that nilotinib rapidly induced CMR in 52.6% of patients who had achieved MMR with continuous detectable BCR-ABL1 levels after long-term imatinib therapy. The safety concerns associated with nilotinib were close to those expected. Cardiovascular events during nilotinib treatment must be managed. Switching to nilotinib 400 mg twice daily may represent an alternative treatment strategy for inducing a deeper response (CMR) in relatively imatinib-resistant CML-CP patients. Disclosures Shibayama: Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kawaguchi:Novartis: Honoraria. Kuroda:Janssen: Honoraria; Astra Zeneca: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Matsumura:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma K.K: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria; Pfizer Japan Inc.: Honoraria. Sunami:Takeda: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Research Funding; Bristol-Myers Squibb K.K.: Research Funding; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding. Akashi:Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Astellas Pharma: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy; Celgene: Research Funding. Kanakura:Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Toyama Chemical: Research Funding; Fujimotoseiyaku: Research Funding.

Description: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Although gene fusions generated through chromosomal translocations, deletions, and inversions are the most frequent genetic abnormalities detected in other types of leukemia, recurrent gene fusions except for SIL-TAL1 have been poorly defined in T-ALL. To discover driver mutations or fusion genes, which involved in the pathogenesis of childhood T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed whole-exome sequencing (WES) and transcriptome sequencing (WTS) in 31 and 46 cases, respectively. We also performed SNP array karyotyping in 46 cases. To detect somatic mutations or fusion transcripts, we used our pipeline "Genomon-exome" and "Genomon-fusion" algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion transcripts were validated by reverse - transcription polymerase-chain-reaction and Sanger sequencing.Recurrent mutations observed in more than 3 cases were detected in NOTCH1 (n = 18, 58%), FBXW7 (n = 7, 23%), PHF6 (n = 5, 16%), GATA3 (n = 4, 13%), MYB (n = 3, 10%), and NRAS (n = 3, 10%), respectively. We identified previously known fusion genes, such as MLL-ENL (n = 2), CALM-AF10 (n = 2), NUP214-ABL1 (n = 1) and FGFROP1-FGFR1 (n = 1). MLL-ENL is one of the frequent translocation in infant multilineage leukemia, but also reported in non-infant B cell precursor ALL and T-ALL. FGFR1OP is ubiquitously expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is thought to promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1. CALM-AF10 leukemia is reported to increase HOXA cluster gene transcription, we could also confirm elevated HOXA genes expression by FPKM value. Four SIL-TAL1 fusions were detected in our cohort. Recently, a novel mutation in TAL1 enhancer region which introduce de novo MYB biding site has been reported. Since this abnormality lead high expression of TAL1, we also analyzed expression data obtained from WTS. Among 46 specimens, 19 samples showed high expression of TAL1 (FPKM value ≥5). In those cases, 4 cases had SIL-TAL1 fusions (8%), and 3 cases (6%) had insertions in enhancer region of TAL1. Subsequent analysis using Gene Set Enrich Analysis (GSEA) between TAL1 high and low expression samples revealed that "LEE_EARLY_T_LYMPHOCYTE_UP" was enriched in TAL1 high expression samples (Enrichment score = 0.73, FDR = 0.073). This gene set includes genes up-regulated at early stages of progenitor T lymphocyte maturation compared to the late stages, and MYB was included in this gene set. Intriguingly, MYB mutation samples were not represented TAL1 high expression. TAL1 related rearrangement or enhancer insertion was not detected in the rest of 12 cases with TAL1 high expression, suggesting that other mechanisms of TAL1 high expression might be exist. In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we also found recurrent MYB mutations. SIL-TAL1 was known as most frequent rearrangement, TAL1 enhancer insertions were also frequent in TAL1 overexpressed samples. TAL1 enhancer insertion and MYB mutation was exclusive, suggesting that TAL1 and MYB have a key role in childhood T-ALL. Consistent with other reports, frequent translocations were not observed in T-ALL, suggesting the genetic differences between T-ALL and other hematological malignancies. Further studies will be necessary to unravel oncogenic mechanisms that implicated in new therapeutic strategy for childhood T-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background: It is important to establish proper intensity of chemotherapeutic regimen for low risk group of childhood B-precursor ALL (BCP-ALL) to avoid late effects. For this purpose, in Japan Association of Childhood Leukemia study (JACLS) ALL-02 clinical trial, we defined standard risk (SR) group as the patients ranged from 1 to 10 years showing white blood cell (WBC) count of less than 10,000/μL at the diagnosis without predonisolone poor responder, t(4;11), t(1;19) and CNS3 to treat with less intensive chemotherapy (JACLS ALL02 SR protocol). Herein, we report the outcome of JACLS ALL02 SR protocol and analyzed prognostic factors to identify super low risk group which is curable by less intensive chemotherapy. Patientss and treatment: JACLS ALL02 SR protocol consisted of early phase therapies for total 4 months including an induction therapy using vincristine (VCR), steroids, pirarubicin (THP-ADR), and L-asparaginase (L-asp) for 4 weeks, a consolidation therapy using cyclophosphamide and a combined administration of cytarabine and dexamethasone (DEX) or 6-mercaptopurine (6-MP), a sanctuary therapy with only two cycles of high-dose methotrexate (MTX) of 3g/m2, one course of 4 drugs re-induction therapy and maintenance phase with 6-MP and oral MTX combined with VCR and PSL every 5 weeks by the end of the second year. In addition, total of 12 times of triple intrathecal therapy were administered by the end of the first year. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Multivariate analysis was performed using a Cox regression model. Results: 1252 newly diagnosed ALL patients were enrolled from April 2002 to May 2008 in JACLS ALL02 clinical trial, which include 388 patients (192 males and 196 females) in SR group (31.0%). The median age at diagnosis was 4.2 years (range, 1.0 to 9.9 years) and the median WBC count was 3,700/μL (range, 370 to 9,984 /μL). The median follow-up time was 6 year and 5 months. 4y / 8y EFS and OS (±SE) of the SR group are 91.5±1.4% / 89.4±1.7% and 97.9±0.7% / 95.2±1.2%, respectively. 4y / 8y EFS of the patients of 1 to 5 years old (n=300) and 6 to 9 years old (n=88) are 93.5±1.4% / 93.0±1.5% and 84.9±3.9% / 75.8±5.8%, respectively (log rank p=0.0003). 4y / 8y EFS of the patients with day 15 M1 marrow (n=287 ) was 92.8±1.6% / 91.6±1.8%, while those of the patients with day 15 M2 marrow (n=101) was 88.0±3.3% / 83.7±3.9% (log rank p=0.0446). When 388 patients were further classified into 4 subgroups based on genetic abnormalities, such as ETV6-RUNX1 (n=89, 22.9%), high hyperdiploid (HHD) (n=97, 25%), normal karyotype (n=147, 37.9%), and others (n=55, 14.2%), 4y-EFS/OS of each group was 97.6±5.9 /100% (ETV6-RUNX1), 91.5±2.9 /100% (HHD), 89.6±2.5 /95.1±1.8% (normal karyotype), and 86.9±4.6 /98.2±1.8% (others), suggesting poor EFS of B-others group. When HHD subgroup was divided into two groups, such as triple trisomy (TT; n=35) (trisomy of chromosome 4,10,17) and non-TT (n=62), 4y-EFS of each sub-group was 100% and 84.5±4.5%, respectively (log rank p=0.0161). Interestingly, day 15 M2 marrow was associated with poor 4y/8y EFS only in the normal karyotype subgroup (day 15 M1 vs M2 92.0±2.6% / 90.2±3.1% vs, 81.3±6.9% / 77.2±7.7%, log rank p=0.0497). The rates for NCI-CTC grade 4 infection, allergy, increase of transaminase were 1.80%, 0.26%, and 22.9%, respectively. Multivariate analysis identifies the patients of 6 to 9 years old (HR=3.178, p=

Description: Background Sustained treatment-free remission (TFR) has been reported in 40-60% of patients with chronic myeloid leukemia-chronic phase (CML-CP) after discontinuation of imatinib or dasatinib following at least 1-2 years of deep molecular response (MR). We investigated safety and efficacy of discontinuing nilotinib treatment after 2 years of sustained MR4.5 (BCR-ABL1IS ≤ 0.0032%) on nilotinib in patient for whom MR4.5 was achieved by prior treatment with imatinib or nilotinib. Methods The Stop Nilotinib (NILSt) trial was a single-arm multicenter phase 2 study in Japan. CML-CP patients who obtained MR4.5 by treatment with imatinib or nilotinib were enrolled, and were further treated with nilotinib for 2 years. The patients who maintained MR4.5 during those 2 years were eligible for discontinuation of nilotinib. After treatment discontinuation, maintenance of MR4.5 was monitored by quantitative RT-PCR every month during the 1st year and every 2 months during the 2nd year. Nilotinib was reintroduced in patients who lost MR4.5. The primary endpoint was the proportion of patients who maintained MR4.5 at 1 year after the discontinuation. This study is registered, number UMIN000007141. Results 112 patients were enrolled between April 11, 2012 and November 30, 2012, and were treated with nilotinib for 2 years. 90 of those patients maintained MR4.5 during the entire 2-year period and were eligible to discontinue treatment, among which 87 patients actually discontinued nilotinib to intend a treatment-free remission period. Median follow-up after the discontinuation was 13.4 months (range 4.8-20.1). At 1 year, 53 patients (58.9%, 90% CI 49.7-67.7) maintained MR4.5, whereas 34 patients experienced loss of MR4.5 mostly within 6 months after the discontinuation (Figure 1). Thirty-two of those 34 patients (94.1%) regained MR4.5 2.2 months (median, 95% CI 1.5-2.6) after reintroduction of nilotinib. The following parameters did not significantly predict the probability of MR4.5 at 1 year after the discontinuation: age, sex, Sokal, Hasford, EUTOS scores, history of IFN-a therapy, total duration of imatinib or nilotinib therapy, time to MR4.5, or trough concentrations of nilotinib in sera. Notably, the percentages of patients maintaining MR4.5 for one year without treatment did not improve significantly with longer duration of prior MR4.5 on treatment; even some patients with a duration of prior deep MR on treatment exceeding 10 years experienced loss of MR4.5 after treatment discontinuation (Table 1). The rates of all grade (grade 3/4 in parentheses) cardiovascular events were 5.5% (2.7%), fluid retention were 14.1% (0%), and musculoskeletal pain were 9.7% (1.8%) during the 2-year treatment periods. Conclusion Nilotinib can be discontinued without relapse in more than half of the patients who maintained MR4.5 for at least 2 years. However, relapse occurred after the discontinuation following even more than 10 years of sustained deep MR in the rest of the patients. This suggests that the period of deep MR after which nilotinib can be discontinued without relapse is considerably long, if any, in a substantial proportion of patients. Biomarkers to detect such patients are awaited. Furthermore, additional strategies may be required to safely discontinue nilotinib as early as possible in such patients, in order to avoid serious adverse events caused by prolonged administration. Figure 1. Kaplan-Meier estimates of TFR after discontinuation of nilotinib Figure 1. Kaplan-Meier estimates of TFR after discontinuation of nilotinib Table 1. Rates of MR4.5 maintenance at 1 year after discontinuation of nilotinib in relation to the duration of deep molecular response before the discontinuation Table 1. Rates of MR4.5 maintenance at 1 year after discontinuation of nilotinib in relation to the duration of deep molecular response before the discontinuation Disclosures Kawaguchi: Novartis: Honoraria. Kuroda:Janssen: Honoraria; Astra Zeneca: Research Funding; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Matsumura:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma K.K: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria; Pfizer Japan Inc.: Honoraria. Kanakura:Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding.