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: 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: 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 Hematopoietic stem cell transplantation (HSCT) is one of the promising treatment strategies for children with refractory Langerhans cell histiocytosis (LCH), because of its immunomodulatory effects.Efficacy and indication of HSCT has been still undetermined. We analyzed the outcomes of HSCT in children with refractory LCH registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation. Patients and methods Between 1996 and 2014, 30 patients

Description: Background: It is well known that PAX5 related fusion proteins are mainly associated with leukemogenesis of B cell precursor acute lymphoblastic leukemia (B-ALL) through dominant negative effect against normal PAX5, resulting in impairment of B cell differentiation. However, different biological property of PAX5 fusion protein which is related to B-ALL has not been fully investigated. Here, we performed functional analysis of novel fusion protein, PAX5-KIDINS220 (P-K220) which was identified in pediatric Ph-like ALL patient. Methods: Full length of P-K220 fusion gene was cloned into pRetroX-Tight-Pur retroviral vector. We also established the PAX5-N construct that contained only PAX5 region (1-306 aa) of P-K220 so that we could analyze the importance of KIDINS220 in P-K220. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing P-K220 or PAX5-N under doxycycline (DOX) dependent manner. P-K220 was also cloned into pAcGFP1-C1 vector and pMSCVneo vector to perform localization assay and luciferase reporter assay. Gene expression analysis was performed using Mouse Genome 430 2.0 Array. In cytotoxic assay using co-culture system with murine mesenchymal stromal cell, MS-5, Ba/F3 cells that were pre-stained with Carboxyfluorescein diacetate succinimidyl ester (CFSE) were seeded on MS-5 stromal cell, then 10 nM vincristine (VCR) was applied. 72 hours later, flow cytometric analysis was performed to determine the fraction and proliferation ability of viable Ba/F3 cells. The proliferation ability was estimated by CFSE fluorescence that attenuates depending on cell proliferation. Results and discussions: P-K220 and PAX5-N were successfully expressed in Ba/F3 cells, confirmed by western blotting. Localization assay revealed that GFP tagged P-K220 was localized in a nucleus of HEK293-T cells under confocal microscopy, suggesting that P-K220 acted as a transcriptional factor. Luciferase reporter assay revealed that P-K220 protein inhibited PAX5 transcriptional activity in dominant negative fashion. Although P-K220 was identified in Ph-like ALL patient and activated JAK2-STAT5 pathway through reduction of Socs5 expression, Ba/F3 cells expressing P-K220 protein did not acquire IL-3 independency. P-K220 attenuated proliferation of Ba/F3 cells (p=0.02), which was in contrast to PAX5-JAK2. To reveal which pathways were affected by P-K220, gene expression analysis was performed. Gene set enrichment analysis revealed that multiple pathways related to chemotaxis, migration, such as IL-15 pathway, were activated in P-K220 expressing Ba/F3 cells. RQ-PCR and western blotting confirmed P-K220 induced expression of IL-15 in RNA (p=0.018) and protein level, suggesting that P-K220 might be associated with infiltration of leukemic cells into extramedullary site. Next, we examined whether P-K220 was associated with sensitivity of chemotherapeutic agents, such as vincristine (VCR), cytarabine, prednisolone, methotrexate, and 6-mercaptoprine. P-K220 expressing Ba/F3 cells showed higher VCR IC50 (0.4 nM vs 2.5 nM) and decreased Annexin V positive fraction under the condition of 10 nM VCR (39.4 % vs 23.8 %, p=0.04). In co-culture analysis with MS-5 stromal cell under the 10 nM VCR, the fraction of viable Ba/F3 cells that adhered to MS-5 was significantly increased with expression of P-K220 than PAX5-N or Dox (-) (11.6% vs 7.3% vs 6.8%, p

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: Introduction: L-asparaginase (L-Asp) is one of the risk factor of thromboembolism during ALL chemotherapy. Although the pathogenesis has been still unclarified, L-Asp may have profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In addition, recent studies demonstrated that the age of over 10-years might be a risk factor for this L-Asp associated coagulopathy. In this study, we hypothesized that change of balance between coagulation and fibrinolysis contributes to hyper-coagulation condition during chemotherapy including L-Asp. In order to clarify our hypothesis, we investigated the global coagulation and fibrinolytic function during the ALL induction therapy with simultaneous thrombin and plasmin generation assay (T/P-GA). Patients: Seventy-two pediatric patients aged 1.0 to 15.2 years (43 males and 29 females) with newly diagnosed ALL were enrolled from Aug. 2014 to Mar. 2018 at four hospitals in Japan. All patients and their families had no thrombotic predisposition. Fifty-five cases (76.4%) (BCP-ALL; n=47, T-ALL; n=7, MPAL; n=1) received BFM-95 oriented protocol consisting of a total of 8 doses of E.coli L-Asp 5,000 U/m2, whilst the others (BCP-ALL; n=13, T-ALL; n=2, MPAL; n=1, Ph-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS)-ALL02-protcol consisting of a total of 6 doses of E.coli L-Asp 6,000 U/m2. Fifty-two cases (72.2%) were categorized to younger group (1-10 years; median, 4.3 years) and the other cases to older group (〉10 years; median, 12.6 years). The percentage of patients of each category were similar in the two treatment protocols (p=0.764). Methods: The global functions of coagulation and fibrinolysis were evaluated using T/P-GA [Matsumoto et al. TH 2013]. This clotting was initiated by the mixture of recombinant human tissue factor (Innovin®, f.c. 1 pM), phospholipid vesicles (f.c. 4 μM) and tissue-type plasminogen activator (f.c. 3.2 nM). Thrombin and plasmin generation were monitored simultaneously using thrombin- and plasmin-specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC and BOC-Glu-Lys-Lys-MAC, respectively) in separate microtiter wells. The first derivatives (velocity) of thrombin and plasmin generation were utilized to derive the parameters, lag time (LT), endogenous potential (EP), peak levels (peak), and time to peak (ttPeak). EP of thrombin generation (T-EP) and plasmin-peak (P-peak) were selected as parameters for evaluation in this study. A ratio of T-EP and P-peak of patients' plasmas to those of control normal plasma were calculated. The conventional laboratory markers of coagulation and fibrinolysis were also monitored by fibrinogen (Fbg), fibrin-Fbg degradation products (FDP), antithrombin (AT), thrombin-AT complex and plasmin-α2 plasmin inhibitor complex. Plasmas were collected at T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post induction phase. Results: None of the cases developed L-Asp associated coagulopathy, and two cases (2.8%) developed induction failure. Thirteen cases (18.1%) received fresh frozen plasma transfusion for low Fbg level, whilst 52 (72.2%) cases received AT supplement for low AT level. Fbg showed a median of 165, 99.0, 97.0 and 316 mg/dl at T0, T1, T2 and T3, respectively, and AT showed a median of 143, 80.9, 80.0 and 105%, respectively, whilst the value of other conventional markers remained within reference value. T-EP revealed a median of 1,134, 1,135, 1,221, 1,277 and 1,102 nM, whilst P-peak showed a median of 5.33, 3.75, 3.63, 4.91 and 5.51 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-peak ratios (Fig. 1). T-EP ratios showed significantly higher at T2 and T3 than at T0 and T1 (p

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.

Description: Background: Acute myeloid leukemia (AML) with RUNX1-RUNX1T1 and CBFB-MYH11 have been recognized as core-binding factor (CBF) AML accounting for 25% of the pediatric AML patients. CBF-AML patients have been considered to have a good prognosis, but 30 - 50 % of the RUNX1-RUNX1T1-positive AML patients experience relapse. This finding suggests that some population of them have risk factors associated with poor outcome. Previous studies revealed that KIT activating mutation as a predictor of poor outcome. In addition, previous studies of relatively small number of patients also revealed that CD56 positivity or CD19 negativity were also poor prognostic factors. However, the relationship between KIT activating mutation and specific pattern of cell surface antigens has not been fully investigated. Aim and methods: We performed a retrospective analysis of RUNX1-RUNX1T1-positive AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol to determine risk factors of relapse using the integration of data including pattern of cell surface markers on leukemic cell at diagnosis, genetic abnormalities, and clinical characteristics. Flow cytometric analysis of immunophenotyping was performed in the central laboratories using same panel of antibodies. Conventional cytogenetic analysis using G-banding was performed as part of the routine work-up. Molecular study using quantitative RT-PCR for the detection of RUNX1-RUNX1T1 and PCR for the detection of FLT3-ITD was also performed as part of the routine work-up. Screening of mutation of 8 genes, such as NRAS, KRAS, KIT, WT1, C/EBPA, ASXL1, ASXL2, and CSF3R, was performed by genomic PCR and Sanger sequencing. A total of 106 AML patients enrolled in the JPLSG AML-05 study were RUNX1-RUNX1T1-positive AML, but we could not obtain the data of cell surface marker in 6 of them. Thus, we analyzed 100/106 (94.3%) patients with RUNX1-RUNX1T1-positive AML. Statistical analysis was performed by Kaplan-Meier method with log-rank test. A Cox proportional hazards model was used to determine risk factors for survival and relapse. Results are reported as adjusted odds ratios with 95% confidence intervals. Statistical significance was set at p 〈 0.05. Results: In entire study population, 8 of the 100 patients died and 24 of the 100 patients experienced relapse, respectively. The 3-year overall survival (OS) and relapse-free survival (RFS) rates were 91.7 % (95 % CI; 83.2 - 96.0) and 69.5 % (95 % CI; 59.0 - 77.9), respectively. In terms of genetic analysis, 21 / 100 (21.0%) patients had KIT exon 17 mutation, 11/100 (11.0%) had KIT exon 8 mutation, 6/100 (6%) had KRAS mutation, 16/100 (16%) had NRAS mutation, 2/100 (2%) had C/EBPA mutation, 2/100 (2%) had WT1 mutation, 6/100 (6%) had ASXL1 mutation, 9/100 (9%) had ASXL2 mutation, and 6/100 (6%) had CSF3R mutation. FLT3-ITD was also identified in 3 of the 100 (3%) patients. In terms of cell surface marker expression pattern, CD19 expression was negative in 59 / 100 (59.0%) patients, and CD56 expression was positive in 43 / 100 (43.0%) patients. Patients with KIT exon 17 mutation were significantly accumulated in the CD19 negative (CD19 (-)) population (18 / 59 vs. 3 / 41, p 〈 0.001). Survival analysis revealed that KIT exon 17 mutation and CD19 (-) were associated with inferior 3y-RFS (KIT exon 17 mutation: negative vs positive, 74.6 vs 50%, p