ALBERT

Description: Introduction Epstein-Barr virus (EBV) is a double-stranded DNA virus that infects 〉95% of the human population and is associated with a substantial risk of cancer development. Most infections in children and adolescents are asymptomatic or result in infectious mononucleosis; however, in some patients, EBV is associated with various hematological malignancies including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), and extranodal NK/T-cell lymphoma. EBV infection is also present in a portion of epithelial cell neoplasms such as gastric cancer and nasopharyngeal carcinoma. Despite the large population risk of cancer associated with EBV, it is poorly understood why only a small subset of EBV-infected individuals develop neoplasms, while others do not. Patients and Methods We designed a target enrichment system to capture several EBV strains including the Akata strain, which is responsible for the majority of EBV infections in Japan. We analyzed the genomes of EBV strains in 139 patients with various EBV-associated diseases and 17 EBV-positive cell lines. Next-generation sequencing reads were aligned to the Akata reference genome to analyze nucleotide variations, copy number alterations, and structural variations including sequence insertions in the human genome. The institutional review board of Nagoya University Graduate School of Medicine approved this study. Results We identified a median of 645 single nucleotide variants (SNVs) in the EBV genomes, 78% of which affected coding sequences. SNVs in coding sequences were significantly biased toward synonymous variants, suggesting negative selection pressure. The SNVs detected in noncoding sequences were enriched in two evolutionarily conserved viral noncoding RNAs (EBER1 and EBER2), particularly in the PAX5-binding domain of EBER2. However, most SNVs identified in the EBV genome do not seem to affect the development of neoplasms, as hierarchical clustering of EBV genomes from neoplastic and non-neoplastic diseases based on SNVs revealed no significant association between the EBV strain and disease type. In addition to SNVs, we identified frequent intragenic deletions in the EBV genomes of patients with EBV-positive DLBCL (10/14, 71%), extranodal NK/T-cell lymphoma (10/23, 43%), chronic active EBV infection (27/77, 35%), and other EBV-associated neoplasms (2/7). Such deletions were also identified in several EBV-associated cell lines (6/17), but not in non-neoplastic diseases such as infectious mononucleosis (0/4) and post-transplant lymphoproliferative disorders (0/14), suggesting a unique role of these mutations in the neoplastic proliferation of EBV-infected cells. Frequent deletions were detected in BamHI A rightward transcripts microRNA clusters (31/156), which suppress viral transcription factors (BZLF1 and BRLF1) required for the lytic reactivation of EBV. Deletions also were associated with several genes essential for virus production (20/156). These observed deletions are thought to upregulate lytic cycle-associated genes, some of which benefit neoplasms by inducing genomic instability and immune escape and mitigate cell damage caused by the production of viral particles. In fact, deletion of one essential gene, BALF5, resulted in upregulation of the lytic cycle and promotion of lymphomagenesis in a xenograft model. Discussion Although the essential roles of several latency-associated genes, such as LMP-1 and EBNA-2, in EBV-mediated immortalization and transformation of human lymphocytes have long been discussed, our finding raises the possibility that lytic cycle-associated genes also contribute to lymphomagenesis. This agrees with reports that lytic cycle-associated genes are expressed in Burkitt lymphoma, DLBCL, and chronic active EBV infection, and that BZLF1-deficient lymphoblastoid cells exhibit significantly impaired tumorigenicity in mice. In addition, essential gene deletions lead to the protection of EBV-infected cells from lysis. Further studies are warranted to exploit these findings for the design of novel therapeutics for EBV-associated neoplasms. Disclosures Kiyoi: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Nakamura:Roche/Chugai,: Research Funding; Kyowa-Kirin: Research Funding.

Description: Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, resistance to ALK inhibitors is a ubiquitous problem in ALK-expressing cell lines as well as treated patients. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. Previous reports have suggested that mechanism of resistance is mediated by mutations in the ALK kinase domain impairing binding of an inhibitor to an ALK protein. Thus, new treatment modalities are urgently needed to sensitize patients to crizotinib thereby improving the management of hematological or solid malignancies harboring ALK mutations. To identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, we implemented a high throughput chemical screen in 4 NB-derived cell lines, using a curated library of ~450 compounds. In the compounds screening, JAK-STAT kinase inhibitor (cucurbitacin I) was the most discriminatory with regard to sensitivity for ALK-mutated cell lines. Since Gamma cytokine JAK/STAT system as a target in the treatment of T-cell or myeloid malignancies, we analyzed the cytotoxicity of cucurbitacin I antitumor effect using an MTT assay revealed cucurbitacin I to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by cucurbitacin I (IC50values:T-ALL lines, ICH-TALL-UK, 103.2 nM; MOLT-14, 35.6 nM; KCMC-T, 134.2 nM; Jurkat, 77 nM; ICH-TALL-SM, 47.7 nM; MOLT-4, 22.2 nM. AML lines, HEL, 66.7 nM; Kasumi-3, 4.5 nM; KG-1, 53.6 nM; THP-1, 337.2 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of cucurbitacin I. In NB cell lines harboring F1174L or R1275Q-mutated ALK, crizotinib combined with cucurbitacin I enhanced tumor responses and showed synergistic cytotoxicity.Although crizotinib and cucurbitacin I alone or combination therapy (cucurbitacin I + crizotinib) did not result in decreased viability over control compared with vehicle, the combination therapy in all of 6 cell lines with ALK aberrations and 10 of 13 ALK wild-type cell lines with MYCN amplification or 11q LOH was more effective than vehicle, crizotinib alone, and cucurbitacin I alone. Analysis of downstream signalling through MAPK, AKT and STAT3 pathways showed that NIH3T3 cells stably expressed F1174L mutated ALK or TGW cells harboring R1275Q-mutated ALK, expressed lower levels of pERK, pAKT and pSTAT3 in combination therapy compared with cells treated with cucurbitacin I or crizotinib alone. These findings may provide a indication that the combination of low-dose ALK and STAT3 inhibitors may be benefitical for the treatment of NB, by enhancing efficacy while reducing toxicity. In conclusion, our studies suggested that NB, T-ALL or AML cell lines also exhibited potent cytotoxic responses to the cucurbitacin I and the combination of ALK and JAK-STAT inhibitors could be a valuable therapeutic option for ALK mutated malignancies such as high-risk NB with potential clinical application. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Empiric antifungal therapy (EAT) is recommended for persistent or recurrent febrile neutropenia based on an old randomized controlled trial, but such treatment is apparently overtreatment for the majority of patients. On the other hand, preemptive therapy triggered by positive blood tests for fungal antigens and/or imaging study findings was shown to increase the incidence of invasive fungal infection, and thus, a risk-based approach is important. The D-index, which is defined as the area over the neutrophil curve during neutropenia and hence reflects both the duration and depth of neutropenia (Figure 1A), enables real-time monitoring of the risk of invasive fungal infection. Previous studies showed that the cumulative D-index (c-D-index), which was calculated as cumulative D-index from the onset of neutropenia (Figure 1B), had high negative predictive values for invasive mold infection or pulmonary infection with cutoff values of 5,800 or 5,500 in high-risk neutropenic patients [J Clin Oncol 2009; 27: 3849-54. Biol Blood Marrow Transplant 2010; 16: 1355-61]. Methods: We investigated a novel approach, called D-index-guided early antifungal therapy (DET) and compared it to EAT in high-risk neutropenic patients. In the EAT group, empiric antifungal therapy was started for persistent (〉=4 days) or recurrent febrile neutropenia. For patients with persistent or recurrent febrile neutropenia in the DET group, preemptive antifungal therapy was applied until c-D-index reached 5,500, but antifungal agent was initiated after c-D-index exceeded 5,500, even if there was no significant finding in serum fungal makers or imaging studies, to prevent excessive invasive fungal infection. Micafungin at 150 mg/day was administered as EAT or DET in this study. We randomized 423 patients who underwent chemotherapy or hematopoietic stem cell transplantation for hematological malignancies, in which predicted period of neutropenia exceeded 7 days, into the EAT group or the DET group, and 413 were eligible for intent-to-treat analyses (201 patients in the EAT group, 212 patients in the DET group). The prophylactic use of fluconazole or itraconazole was allowed. Primary endpoint was the development of proven/probable invasive fungal infection. Results: Backgrounds of the patients were similar between the 2 groups (Table 1). Invasive fungal infection (proven/probable/possible) was observed in 12 patients (6.0%) of the EAT group and 5 patients (2.4%) of DET group, respectively. Proven/probable invasive fungal infection was identified in 5 patients (2.5%) of the EAT group and 1 patient (0.5%) of DET group, which fulfilled the predetermined criteria of non-inferiority of the DET group. Regarding the pathogens, the EAT group included 1 case of candidemia and 4 cases of invasive pulmonary aspergillosis, and the DET group included one fusariosis. The survival rate of the EAT and DET group was 98.0% vs. 98.6% at day 42 and 96.4% vs. 96.2% at day 84, respectively. During the observation period, 31 patients died due to disease progression (n=19), infection (n=5) or other causes (n=7). Causes of infection related mortality included Pseudomonas aerginosa infection (n=2), fusariosis (n=1), toxoplasmosis (n=1) and septic shock by unknown pathogen (n=1). The frequency of micafungin use was significantly lower in the DET group than the EAT group (32.5% vs. 60.2%, P

Description: Aberrant DNA methylation profiles in various types of cancer have highlighted the importance of DNA methylation in human carcinogenesis and opened the prospect of targeting aberrant DNA methylation with demethylating agents as a therapy for cancer. Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy derived from CD4 (+) T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Although it has been estimated that 20 million people are infected with HTLV-1 worldwide, most HTLV-1 infected individuals have no symptoms and only 3-7% of HTLV-1 positive carriers develop ATL in their life time. HTLV-1 infected cells undergo multistep leukemogenesis and there are four clinical subtypes of ATL, smoldering type, chronic type, acute type, and lymphoma type. It takes several decades to acquire aggressive phenotypes through the accumulation of genetic and epigenetic abnormalities. In this study, we aimed to elucidate the contribution of aberrant DNA methylation to ATL leukemogenesis and the anti-ATL effects of DNA demethylating agents. Since the expression profiles of CADM1 and CD7 in CD4 (+) T-cells reflect ATL disease progression, fractions of HTLV-1 infected cells and normal T-cells were isolated from ATL patients, HTLV-1 carriers, and healthy volunteers using the expression statuses of CADM1 and CD7. Comprehensive genome-wide profiling of DNA methylation was performed by quantitative array-based methylation analysis at the single-CpG-site level using the Infinium HumanMethylation450 BeadChip array. Anti-ATL effects of four DNA demethylating agents were investigated in in vitro experiments using ATL-related cell lines and a xenograft mouse model. Unsupervised hierarchical clustering analysis was first conducted using the DNA methylation profiles of 20,000 CpG probes, which were randomly picked from 470,870 probes. Global DNA hypomethylation was detected in HTLV-1 infected cells at the asymptomatic carrier stage. To identify differentially methylated positions (DMPs) that specifically reflect ATL disease status, the DNA methylation profiles of a normal cell subpopulation were compared with those of a HTLV-1 infected subpopulation. We identified 12,025 hypermethylated and 33,581 hypomethylated DMPs that were specific to the HTLV-1 infected subpopulation. Importantly, the methylation profiles of hypermethylated DMPs, but not those of hypomethylated DMPs, were different between the aggressive and indolent types of ATL and could be used to distinguish them. Therefore, we next extracted and analyzed 1,207 hypermethylated CpG sites located in TSS200 CpG islands (CGIs) in hypermethylated DMPs, since TSS200 CGIs are widely recognized to regulate gene expression in a methylation dependent manner. We found the DNA methylation profiles of 1,207 probes tended to correlate with ATL disease status. Since regional DNA hypermethylation appears to be associated with ATL disease progression, we tested the anti-leukemia activities of two novel decitabine prodrugs, OR-21 and OR-12, and compared their efficacies with those of clinically available AZA and DAC. OR-21 and OR-12 showed enhanced oral bioavailability and sustained release of decitabine and decitabine 5'-monophosphate, respectively. In ATL-related T-cell lines cultured in the presence of each DNA demethylating compound, DAC and OR-21 significantly suppressed cell growth and decreased DNA methylation at LINE-1 repeat regions, which are used as a biomarker for the monitoring of global changes in DNA methylation. To establish a xenograft mouse model, cells of the HTLV-1-transformed T-cell line, MT-2, were inoculated into the subcutaneous tissue of immunodeficient Balb/c Rag-2-/- Jak3-/- mice, which lack mature T and B lymphocytes and NK cells. A visible tumor appeared 10 days after inoculation and DAC or OR-21 was injected intraperitoneally twice a week, because they were rapidly degraded in the stomach acidic environment and could not be administered orally. The AUC-guided dosing of OR21 (3.39mg/kg) suppressed tumor growth, which was comparable with that obtained with 1.25 mg/kg DAC, while OR21 showed less hematotoxicity than DAC. Disclosures Watanabe: OHARA Pharmaceutical Co., Ltd.: Research Funding. Ureshino:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Fukuda-Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Kimura:OHARA Pharmaceutical Co., Ltd.: Research Funding.

Description: Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing 〈 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P

Description: Background : Anagrelide is a widely used therapeutic agent for patients with essential thrombocythemia. While other cytoreductive agents, such as hydroxyurea, influence multi-lineage blood cells, anagrelide exerts less effect on the white and red blood cell lineages. Although the clinical efficacy of anagrelide has been reported, the exact mechanism of action is unclear. Recently, immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from human induced pluripotent stem (iPS) cells by the introduction of doxycycline-inducible lentiviral vectors harboring c-MYC, BMI1, and BCL-XL for the clinical application of artificially generated platelets. In this study, we aimed to elucidate the molecular mechanism of anagrelide on the inhibition of platelet production using imMKCLs as an ideal model for human megakaryogenesis and platelet formation. Materials and Methods : imMKCLs, established at Center for iPS Cell Research and Application, Kyoto University, Japan, were cultured in Iscove's modified Dulbecco's medium with thrombopoietin (TPO), stem cell factor (SCF), and doxycycline. The differentiation of imMKCLs and platelet generation were induced by doxycycline removal. The generation of mature platelets was observed approximately 7 days after the differentiation was initiated. Both undifferentiated and differentiated imMKCLs were treated with several different concentrations of anagrelide. The cell proliferation and number of generated platelets following anagrelide treatment were analyzed by BrdU cell proliferation assay and flow cytometry, respectively. To explore the molecular mechanism of anagrelide treatment in imMKCLs, we performed mRNA sequencing in imMKCLs treated with or without anagrelide followed by gene ontology (GO) analysis and gene set enrichment analysis (GSEA). The expression of genes related to megakaryogenesis and platelet formation was also analyzed utilizing quantitative real-time PCR. Results : Anagrelide exposure caused morphologically suppressive changes in the differentiation of imMKCLs. Anagrelide treatment also suppressed the mRNA expression of the megakaryocytic surface markers CD41 and CD61 in both undifferentiated (P 〈 0.01 and P 〈 0.001, respectively) and differentiated (P 〈 0.01 and P 〈 0.001, respectively) settings. The BrdU incorporation rate in differentiated imMKCLs decreased significantly following anagrelide treatment (P 〈 0.001, anagrelide 0 vs. 1 or 10 µM). The resultant generation of mature platelets (double positive for CD41 and CD42b) was significantly decreased by exposure to anagrelide, as analyzed by flow cytometry (P 〈 0.001). Regarding the molecular mechanism of anagrelide treatment on imMKCLs, GO analysis following RNA sequencing demonstrated that gene sets related to platelet activation and degranulation were significantly downregulated in both undifferentiated and differentiated conditions. Moreover, GSEA revealed that gene sets related to the cell cycle, such as mitosis and DNA replication, were decreased as well as platelet-specific genes. The mRNA expression levels of genes related to megakaryogenesis and platelet-formation, such as FLI1, TAL1, GATA1, and PF4, were significantly downregulated, especially in differentiated imMKCLs, by anagrelide treatment (P 〈 0.001, P = 0.013, P 〈 0.01, and P 〈 0.01, respectively). Conclusions : We successfully reproduced the platelet-lowering effect of anagrelide by using imMKCLs from human iPS cells that could generate functional platelets in culture. Our RNA sequencing results revealed that anagrelide specifically suppressed megakaryogenesis and platelet formation-related genes. Additional studies including an apoptosis assay and cell cycle analysis of imMKCLs following anagrelide exposure are ongoing to elucidate further molecular mechanisms of anagrelide treatment. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding. Eto:Megakaryon co. Ltd.: Research Funding.

Description: Background and Aim: Treatment with a tyrosine kinase inhibitor (TKI) is the standard of care for patients with chronic myeloid leukemia (CML). Based on the results of the ENESTnd and DASISION studies, the European LeukemiaNet 2013 guidelines now recommend the use of TKIs, imatinib (400 mg once daily), nilotinib (300 mg twice daily), and dasatinib (100 mg once daily) as first-line treatment for patients with CML in the chronic phase (CML-CP). Compared to imatinib, the new generation TKIs, nilotinib and dasatinib, were found to have deeper and faster treatment response rates with acceptable toxicities. However, a direct comparison between nilotinib and dasatinib has never been reported previously. Our study aims to compare the outcomes and molecular responses achieved following the first-line use of these two agents in patients with CML-CP. Patients and Methods: The database of the CML Cooperative Study Group, which includes patients who were initially diagnosed with CML after the introduction of imatinib (April 2001), was reviewed, and patients who were given nilotinib or dasatinib as first-line therapy were identified. The event-free survival (EFS, defined as the loss of treatment efficacy, disease progression, or any cause of death), and rates of cumulative major molecular response (MMR), and deep molecular response (DMR, defined as the depth of MR 4.5) were compared between the nilotinib and dasatinib groups. Further, the predictive ability of the Sokal, Hasford, and European Treatment and Outcome Study (EUTOS) scoring systems for the achievement of molecular responses was also evaluated. For the analysis of molecular responses, patients who switched from their initial treatment agent to another TKI before achievement of MMR or DMR were considered to have no MMR or DMR. Results and Discussion: Out of 361 patients with CML-CP enrolled in our database, 58 and 63 were treated with conventional doses of nilotinib (300 mg twice daily) and dasatinib (100 mg once daily), respectively, as first-line therapy. Patients who had been started on a low dose TKI therapy were excluded from this analysis. The patient demographics, including age, sex, observation periods, and the Sokal, Hasford, and EUTOS scores did not show significant differences between the groups. In total, there were five events during the observation period (1 in the nilotinib group and 4 in the dasatinib group), and all events were deaths unrelated to CML, except for one in a patient in the dasatinib group who showed loss of complete cytogenetic response. The disease did not progress to the accelerated or blastic phase in any of the cases. The EFS did not differ between these two groups (p = 0.214). The MMR rates by 6, 12, 18, and 24 months were 59%, 72%, 74%, and 81%, respectively, in the nilotinib group and 52%, 73%, 81%, and 86%, respectively, in the dasatinib group. The DMR rates by 6, 12, 18, and 24 months was 7%, 17%, 24%, and 28%, respectively, in the nilotinib group and 3%, 16%, 25%, and 29%, respectively, in the dasatinib group. During the first 24 months of treatment, 4 (7%) patients in the nilotinib group and 11 (17%) in the dasatinib group had been switched to other TKIs (p = 0.0983). Among the three scoring systems, only the Hasford score could predict the achievement of DMR, and all of them failed to predict the achievement of MMR in the entire cohort. Our data suggest that both nilotinib and dasatinib have comparable efficacies, with high molecular response rates and promising outcomes. The validity of our findings should be tested in a randomized study, which is currently underway in Japan. Disclosures Takaku: Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Speakers Bureau. Tokuhira:Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Chugai: Speakers Bureau. Asou:Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Asahi Kasei Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Kizaki:Nippon Shinyaku,: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Speakers Bureau. Kawaguchi:Novartis Pharma K.K.: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Alexion: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Description: Introduction Myelofibrosis (MF) occurrence can be attributed to various pathogenic mechanisms. A recent study showed that the neoplastic clone of fibrocytes (spindle shaped, fibroblast-like blood cells derived from monocyte lineage) was essential in primary MF pathogenesis; moreover, serum amyloid P (PRM-151), which suppresses fibrocyte differentiation, markedly improved survival and MF in a murine xenograft model (J Exp Med 2016; 213: 1723). Using a romiplostim-induced murine MF model, direct induction of fibrocyte differentiation using TPO receptor activation, leading to MF progression, was demonstrated (Leukemia 2017; 31: 2709). Using DNA microarray analysis, we previously showed that compared with macrophages, human fibrocytes highly express SLAMF7 (J Immunol 2015; 195: 4341). Elotuzumab (Elo) is an anti-SLAMF7 antibody recently developed for treating multiple myeloma. We showed that Elo inhibits human fibrocyte differentiation in vitro and relieves MF using the mouse model in vivo, identifying Elo as a potential therapeutic agent (manuscript in preparation). Additionally, monocytes with high SLAMF7 expression and negative CD16 expression were significantly elevated in the peripheral blood (PB) of MF patients compared with that of healthy controls (HCs). We hypothesized that this monocyte subset reflects fibrocyte activation and is the target of Elo. For the clinical study of Elo, we evaluated SLAMF7high CD16− monocyte percentage in PB of HCs, myeloproliferative neoplasm (MPN) patients with or without MF, and non-MPN patients with MF in a cross-sectional manner. Methods Six HCs, 12 non-MPN patients with MF, and 44 MPN patients (18 without and 26 with MF) were enrolled; their blood samples were collected. All MPN patients underwent bone marrow biopsy in 2016-2018 and were diagnosed by the 2016 WHO classification and diagnostic criteria. Non-MPN patients underwent bone marrow biopsy in 2015-2018, and MF was confirmed over MF-1 according to the European Consensus Criteria. SLAMF7high CD16- monocyte percentage in PB monocytes of HCs and patients was calculated using flow cytometry. Further, all patients were tested for JAK2V617F, CALR, or MPL genetic mutations. The allele burden of JAK2V617F was measured. Moreover, fibrocytes were differentiated from PB mononuclear cells of patients with genetic mutation and were verified for genetic mutation in their fibrocytes. Results Patients were classified in three groups (MPN without MF, MPN with MF, and non-MPN with MF). The median percentage of SLAMF7high CD16− monocytes in PB of HCs, MPN patients without MF, MPN patients with MF, and non-MPN patients with MF were 1.66%, 2.48%, 27.4%, and 19.8%, respectively. Compared with HCs and MPN patients without MF, SLAMF7high CD16− monocyte percentage of MPN and non-MPN patients with MF significantly increased (p 〈 0.05 and p 〈 0.01, respectively) (Figure A). MPN patients with MF harboring JAK2V617F (n = 17) had a significantly higher SLAMF7high CD16− monocyte percentage than those without MF harboring JAK2V617F (n = 8) (median: 43.70% vs. 7.00%, p 〈 0.001). While a similar trend was observed in patients not harboring JAK2V617F (median: 1.15% vs. 3.26%, p = 0.05), the contrast between patients with and without MF patients was significant for those with JAK2V617F (Figure B). More than 25% of SLAMF7high CD16− monocytes in PB indicates MF in MPN patients harboring JAK2V617F (sensitivity: 82.4%, specificity: 87.5%). In addition, the JAK2V617F allele burden of fibrocytes was strongly correlated with the SLAMF7high CD16− monocyte percentage in PB (p = 0.0003) (Figure C). In all patients, fibrocytes and PB harbored the same genetic mutation. Conclusion The percentage of SLAMF7high CD16− monocytes in PB elevated in MF patients regardless of MPN, whereas it was not elevated in MPN patients without MF. In MPN patients, those with MF harboring JAK2V617F presented a high SLAMF7high CD16-monocyte percentage in PB, positively correlating with the JAK2V617F allele burden of fibrocytes. In conclusion, SLAMF7high CD16- monocyte levels are closely related with MF and neoplastic clones of fibrocytes harboring JAK2V617F, indicating fibrocyte activation and representing a non-invasive marker of MF progression and a potential target for Elo treatment. Figure. Figure. Disclosures Usuki: Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Novartis: Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau.

Description: [Background] CD34-positive monocytes (CD34+mono) have recently been identified following mobilization by granulocyte-colony stimulating factor (G-CSF), and have been suggested to have a potential to modulate immune functions in animal models. However, the biological feature of CD34+mono in humans still remains unclear. Thus, we explored the difference between CD34+mono, CD34+cells, and monocytes through the analyses of gene expression profiles (GEP). [Methods] CD34+mono　(Lin-CD34+CD33+CD14+CD11b+, Figure1), CD34+cells (Lin-CD34+CD33-CD14-CD11b- ), and monocytes (Lin-CD34-CD33+CD14+CD11b+) were directly sorted into tubes from cryopreserved grafts from three healthy donors. After the extraction of total RNA, microarray analyses were performed with GeneChip™ 3' IVT Pico Kit and analyzed according to the algorithm of Transcriptome Analysis Console (Thermo Fisher Scientific). Condition false discovery rate (FDR) F-test was used for filtering genes, and the threshold was

Description: Introduction NOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations. Methods A total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS). Results In total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern. Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors. Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations. Conclusions We observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. Disclosures No relevant conflicts of interest to declare.