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PDF file - 160K, FISH findings for interphase nuclei

PDF file - 195K

PDF file - 108K, Knockdown of PVT1-NBEA chimeric transcript using siRNA

PDF file - 243K, Genome copy number changes at 8q24 detected by SNP array analysis

PDF file - 467K, Genome copy number alterations of translocation partner loci

PDF file - 91K, MYC and PVT1 expression in cell lines and normal lymphocytes

The prognosis of pediatric acute myeloid leukemia (AML) has improved via stratification therapy. However, relapse or death occurs in 30%-40% of cases. Novel genetic factors for pediatric AML need to be elucidated to improve prognosis. We detected recurrent internal tandem duplication in upstream binding transcription factor (UBTF-ITD) in 1.2% (6/503) of Japanese pediatric patients with de novo AML. No UBTF-ITD was detected in 175 adult patients with AML or in 65 cell lines that included 15 AML, 39 acute lymphoblastic leukemia, five chronic myeloid leukemia, and six neuroblastoma cell lines. All UBTF-ITDs were found in exon 13 and shared a duplicated region. UBTF-ITD was more frequently detected in patients with trisomy 8, FLT3-ITD, WT1 mutation, and/or high PRDM16 expression (trisomy 8, 3/6; FLT3-ITD, 5/6; WT1 mutation, 2/6; and high PRDM16 expression, 6/6). Gene expression patterns of patients with UBTF-ITD were similar to those of patients with NUP98::NSD1 or FUS::ERG. Survival analysis of the AML-05 cohort revealed that patients with UBTF-ITD had worse outcomes than those without UBTF-ITD (3-year event-free survival, 20% vs. 55%; 3-year overall survival, 40% vs. 74%). Moreover, among the 27 patients with trisomy 8, all three patients with UBTF -ITD had a poor prognosis resulting in early events (relapse or non-complete remission) within 1 year. Our findings suggest that UBTF-ITD may be a novel and significant prognostic factor for pediatric patients with AML.

Myeloid leukemia of Down syndrome (ML-DS) is associated with good response to chemotherapy, resulting in favorable outcomes. However, no universal prognostic factors have been identified to date. To clarify a subgroup with high risk of relapse, the role of minimal residual disease (MRD) was explored in the AML-D11 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group. MRD was prospectively evaluated at after induction therapy and at the end of all chemotherapy, using flow cytometry (FCM-MRD) and GATA1-targeted deep sequencing (GATA1-MRD). A total of 78 patients were eligible and 76 patients were stratified to the standard risk (SR) group by morphology. In SR patients, FCM-MRD and GATA1-MRD after induction were positive in 5/65 and 7/59 patients, respectively. Three-year event-free survival (EFS) and overall survival (OS) rates were 93.3% and 95.0% in the FCM-MRD-negative population, and 60.0% and 80.0% in the positive population. Three-year EFS and OS rates were both 96.2% in the GATA1-MRD-negative population, and 57.1% and 71.4% in the positive population. Adjusted hazard ratios for associations of FCM-MRD or GATA1-MRD with EFS were 10.98 (p = 0.01) and 27.68 (p < 0.01), respectively. Detection of MRD by either FCM or GATA1 after initial induction therapy represents a significant prognostic factor for predicting ML-DS relapse.

For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.

The prognosis for infants with acute lymphoblastic leukemia (ALL), particularly those with KMT2A gene rearrangement (KMT2A-r), is dismal. Continuous efforts have been made in Japan to investigate the role of hematopoietic stem cell transplantation (HSCT) for infants with KMT2A-r ALL, but improvement in outcome was modest. In the Japanese Pediatric Leukemia/Lymphoma Study Group MLL-10 trial, infants with ALL were stratified into 3 risk groups (low risk [LR], intermediate risk [IR], and high risk [HR]) according to KMT2A status, age, and presence of central nervous system leukemia. Children’s Oncology Group AALL0631 modified chemotherapy with the addition of high-dose cytarabine in early intensification was introduced to KMT2A-r patients, and the option of HSCT was restricted to HR patients only. The role of minimal residual disease (MRD) was also evaluated. Ninety eligible infants were stratified into LR (n = 15), IR (n = 19), or HR (n = 56) risk groups. The 3-year event-free survival (EFS) rate for patients with KMT2A-r ALL (IR + HR) was 66.2% (standard error [SE], 5.6%), and for those with germline KMT2A (KMT2A-g) ALL (LR), the 3-year EFS rate was 93.3% (SE, 6.4%). The 3-year EFS rate was 94.4% (SE, 5.4%) for IR patients and 56.6% (SE, 6.8%) for HR patients. In multivariable analysis, female sex and MRD ≥0.01% at the end of early consolidation were significant factors for poor prognosis. Risk stratification and introduction of intensive chemotherapy in this study were effective and were able to eliminate HSCT for a subset of infants with KMT2A-r ALL. Early clearance of MRD seems to have translated into favorable outcomes and should be incorporated into risk stratifications in future trials. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) as #UMIN000004801.

B-Cell leukemia/lymphoma 11B (BCL11B) is a transcription factor important for T-cell development and acts as a tumor suppressor gene in T-cell acute lymphoblastic leukemia. Here, we identified BCL11B as a candidate leukemia-associated gene in human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma (ATLL). Interestingly, the short form lacking exon 3 (BCL11B/S) protein was more highly expressed than the full-length BCL11B (BCL11B/L) in leukemic cells from most of the ATLL patients, although expression ratios of BCL11B/L to BCL11B/S were almost equal in control CD4

Recent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, ∼40% of patients with pediatric AML relapse, resulting in a relatively low overall survival rate of ∼70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. We performed transcriptome analysis (RNA sequencing [RNA-seq]) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial and investigated correlations between genetic aberrations and clinical information. Using RNA-seq, we identified 54 in-frame gene fusions and 1 RUNX1 out-of-frame fusion in 53 of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through reverse transcription polymerase chain reaction and RNA-seq. Five gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, MLLT10-DNAJC1, TBL1XR1-RARB, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, ETV6-CTNNB1, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially disease-causing alterations in nearly all patients with AML, including novel gene fusions. Our results indicated that a subset of patients with pediatric AML represent a distinct entity that may be discriminated from their adult counterparts. Based on these results, risk stratification should be reconsidered.

Although infants (age

RAS pathway alterations have been implicated in the pathogenesis of various hematological malignancies. However, their clinical relevance in pediatric acute myeloid leukemia (AML) is not well characterized. We analyzed the frequency, clinical significance, and prognostic relevance of RAS pathway alterations in 328 pediatric patients with de novo AML. RAS pathway alterations were detected in 80 (24.4%) out of 328 patients: NF1 (n = 7, 2.1%), PTPN11 (n = 15, 4.6%), CBL (n = 6, 1.8%), NRAS (n = 44, 13.4%), KRAS (n = 12, 3.7%). Most of these alterations were mutually exclusive and were also mutually exclusive with other aberrations of signal transduction pathways such as FLT3-ITD (p = 0.001) and KIT mutation (p = 0.004). NF1 alterations were frequently detected in patients with complex karyotype (p = 0.031) and were found to be independent predictors of poor overall survival (OS) in multivariate analysis (p = 0.007). At least four of seven patients with NF1 alterations had bi-allelic inactivation. NRAS mutations were frequently observed in patients with CBFB-MYH11 and were independent predictors of favorable outcomes in multivariate analysis [OS, p = 0.023; event-free survival (EFS), p = 0.037]. Patients with PTPN11 mutations more frequently received stem cell transplantation (p = 0.035) and showed poor EFS than patients without PTPN11 mutations (p = 0.013). Detailed analysis of RAS pathway alterations may enable a more accurate prognostic stratification of pediatric AML and may provide novel therapeutic molecular targets related to this signal transduction pathway.

Myeloid sarcoma (MS) is a rare neoplastic condition that is often described in association with acute myeloid leukemia (AML). MS in childhood has received little attention, particularly in Japan. We carried out a nationwide retrospective analysis of Japanese children diagnosed with MS without bone marrow involvement. Inclusion criteria were diagnosis of MS at younger than 20 years of age between January 1, 2000 and December 31, 2013. There was a predominance of males (8:2), and the median age at MS diagnosis was 4 years. Sites of involvement varied and included skin (n = 3), head and/or neck (n = 2), and multiple sites (n = 2). Karyotypes were evaluated in seven patients, with one individual carrying t(8;21) and t(9;11). Four patients developed bone marrow involvement 2-55 months after diagnosis of MS. All patients received chemotherapy for de novo AML and two individuals received HSCT in first remission. Seven of ten patients survived for 50-152 months (median, 93 months) without disease after initial chemotherapy. This retrospective study confirmed that pediatric MS without bone marrow involvement in Japan is a very rare disease. MS patients responded favorably to therapies for de novo AML, and HSCT in first remission was not indicated for all patients.

Therapy-related leukemia (t-leukemia) is associated with dismal prognosis. Published pediatric t-leukemia data are somewhat outdated and may not reflect recent advances in treatment. We report a retrospective nationwide survey of patients diagnosed between 2000 and 2013 in Japan. We identified 43 patients with pediatric t-leukemia; 33 had t-acute myeloid leukemia (t-AML), eight had t-acute lymphoblastic leukemia (t-ALL) and two had t-acute undifferentiated leukemia. Median age at onset and latency were 12 years and 3.8 years, respectively, consistent with previous reports. Of t-AML patients, 63.6% harbored topoisomerase II inhibitor (topo II)-related genetic abnormalities, while only 12.5% of t-ALL patients had such alterations, suggesting that topo II is not key to t-ALL leukemogenesis. The 7-year overall survival (OS) for all 43 patients was 39.2 ± 11.6%. The 5-year OS was 50 ± 20.4% in t-ALL, and 55.2 ± 11.0% in t-AML. Allogeneic hematopoietic cell transplantation (allo-HCT) was associated with superior 5-year OS (HCT(+) vs. HCT(−), 78.8 vs. 12.1%; p

We have experienced 3 consecutive cases of familial hemophagocytic lymphohistiocytosis (FHL). All affected infants had mutations in exon 3 of the perforin gene. The first had a homozygous mutation, c.1168C>T (p.R390*), caused by maternal uniparental isodisomy. The second and third had compound heterozygous mutations: c.781G>A (p.E261K) and c.1491T>A (p.C497*); c.1724G>T (p.C242G) and p.R390*, respectively. FHL is very rare in Northern Japan but should be suspected if infants exhibit prolonged fever. This is the first report of a relationship of p.R390* with FHL caused by uniparental isodisomy, and the second reported case of FHL type 2 with this form of inheritance.

Cytogenetic/molecular heterogeneity is the hallmark of multiple myeloma (MM). However, we recently showed that the serine/threonine kinase PDPK1 and its substrate RPS6KA3 (also termed RSK2) are universally active in MM, and play pivotal roles in myeloma pathophysiology. In this study, we assessed involvement of aberrant miR-375 repression in PDPK1 overexpression in MM. An analysis of plasma cells from 30 pre-malignant monoclonal gammopathies of undetermined significance and 73 MM patients showed a significant decrease in miR-375 expression in patient-derived plasma cells regardless of the clinical stage, compared to normal plasma cells. Introduction of miR-375 reduced PDPK1 expression in human myeloma cell lines (HMCLs), indicating that miR-375 is the dominant regulator of PDPK1 expression. In addition, miR-375 introduction also downregulated IGF1R and JAK2 in HMCLs. CpG islands in the MIR375 promoter were pathologically hypermethylated in all 8 HMCLs examined and in most of 58 patient-derived myeloma cells. Treatment with SGI-110, a hypomethylating agent, and/or trichostatin A, a histone deacetylase inhibitor, increased miR-375 expression, but repressed PDPK1, IGF1R and JAK2 in HMCLs. Collectively, these results show the universal involvement of overlapping epigenetic dysregulation for abnormal miR-375 repression in MM, which is likely to contribute to myelomagenesis and to subsequent myeloma progression by activating oncogenic signalling pathways.

Pediatric acute megakaryoblastic leukemia in non-Down syndrome (AMKL) is a unique subtype of acute myeloid leukemia (AML). Novel CBFA2T3-GLIS2 and NUP98-KDM5A fusions recurrently found in AMKL were recently reported as poor prognostic factors. However, their detailed clinical and molecular characteristics in patients treated with recent improved therapies remain uncertain. We analyzed molecular features of 44 AMKL patients treated on two recent Japanese AML protocols, the AML99 and AML-05 trials. We identified CBFA2T3-GLIS2, NUP98-KDM5A, RBM15-MKL1, and KMT2A rearrangements in 12 (27%), 4 (9%), 2 (5%), and 3 (7%) patients, respectively. Among 459 other AML patients, NUP98-KDM5A was identified in 3 patients, whereas CBFA2T3-GLIS2 and RBM15-MKL1 were only present in AMKL. GATA1 mutations were found in 5 patients (11%). Four-year overall survival (OS) and event-free survival (EFS) rates of CBFA2T3-GLIS2-positive patients in AMKL were 41.7% and 16.7%, respectively. Three-year cumulative incidence of relapse in CBFA2T3-GLIS2-positive patients was significantly higher than that of CBFA2T3-GLIS2-negative patients (75.0% vs. 35.7%, P = 0.024). In multivariate analyses, CBFA2T3-GLIS2 was an independent poor prognostic factor for OS (HR, 4.34; 95% CI, 1.31-14.38) and EFS (HR, 2.95; 95% CI, 1.20-7.23). Furthermore, seven (54%) of 13 infant AMKL patients were CBFA2T3-GLIS2-positive. Notably, out of 7 CBFA2T3-GLIS2-positive infants, six (86%) relapsed and five (71%) died. Moreover, all of CBFA2T3-GLIS2-positive patients who experienced induction failure (n = 3) were infants, indicating worse prognosis of CBFA2T3-GLIS2-positive infants. These findings indicated the significance of CBFA2T3-GLIS2 as a poor prognostic factor in AMKL patients, particularly in infants.

Introduction: Myeloid/Natural killer (NK) cell precursor acute leukemia (MNKPL) is a rare hematologic malignancy prevalent in East Asia. MNKPL is characterized by extramedullary involvement, immature lymphoblastoid morphology without myeloperoxidase (MPO) reactivity, the CD7+/CD33+/CD34+/CD56+/HLA−DR+ phenotype. MNKPL is classified as mixed phenotype acute leukemia, and not otherwise specified rare types (MPAL NOS rare types) in WHO classification. However, its characteristic clinical feature and undetermined genetic feature suggests that MNPKL leaves open the possibility of a new independent disease concept. Here, we report clinical features and genetic alterations in patients with MNKPL. Methods: The Leukemia and Lymphoma Committee of the Japanese Society of Pediatric Hematology and Oncology (JSPHO) sent out questionnaires to 110 JSPHO affiliated hospitals and collected cases of MNPKL diagnosed during the period 2000-2013. Besides, the cases published as literature were recruited. The data of clinical features, cell surface antigen profiling, overall survival (OS), and event-free survival (EFS) defined as relapse or death were also collected as a secondary survey. The protocol of this retrospective study was approved by the review boards of JSPHO and Ehime Prefectural Central Hospital. Comprehensive genetic analysis including 13 whole-exome sequences (WES), 2 target sequence, 6 RNA sequence (RNA-seq), and 8 DNA methylation analysis was performed. We also performed single-cell RNA-seq using 1 sample of MNKPL patients and a normal bone marrow sample as the reference. The research protocol was approved by the review board of TMDU. Results: Sixteen children or young adults (< 39 years old) and 2 older adults with MNKPL were identified. The median age of MNKPL patients was 11 (0.5-75) years old. There are 12 males and 6 females. The extramedullary involvement was observed in 7 patients. Complete remission after induction therapy was achieved in 8/14 (57%) patients treated with acute myeloid leukemia (AML) type chemotherapy and 2/4 (50%) patients treated with acute lymphoblastic leukemia (ALL)/non-Hodgkin lymphoma type chemotherapy, respectively. Fifteen patients underwent hematopoietic cell transplantation (HCT). The median follow-up period was 3.8 (0.1-16.0) years. 5-year OS and 5-year EFS was 49.5% and 40.7%, respectively. In genetic analysis, median 388 somatic mutations in MNKPL were identified by WES. The recurrent mutations were observed in NOTCH1 (n=5), MAML3 (n=4), NRAS, MAP3K4, RECQL4, CREBBP, ASXL2, and KMT2D (n=3, respectively), and MAML2, MAP3K1, FLT3, CARD11, MSH4, FANCI, WT1, ZNF384, and ERG (n=2, respectively). The distinct expression pattern, higher expression of RUNX3 and NOTCH1, and lower expression of BCL11B were identified in MNKPL samples which were compared to MPAL, AML, and T cell ALL in RNA-seq. The distinct methylation profile, hypomethylation of RUNX3 regulatory region, and hypermethylation of BCL11B regulatory region were identified in DNA methylation analysis. Single-cell RNA-seq analysis also showed distinct 4 subsets of MNKPL. Discussion and Conclusions: NK cells are the founding member of a family of innate lymphoid cells (ILC). Genetic abnormality of NOTCH1 pathway is a hallmark of MNPKL. RUNX3 is required for NK cell survival and proliferation in response to IL-15 signaling. RUNX3 high expression and hypomethylation of RUNX3 regulatory region also characterize MNKPL. Currently, MNKPL is classified as MPAL NOS, our genetic analysis revealed that MNKPL is a distinct group from MPAL. The prognosis of MNKPL was not satisfactory even though HCT was performed. The development of new therapeutic approaches based on these genetic analyses is highly expected. Disclosures Saito: Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

Expression of intragenic exon rearrangements (IERs) has reportedly been detected in both normal and cancer cells. However, there have been few reports of occurrence of these rearrangements specific to neoplasms including malignant lymphoma. In this study, we detected IERs of ten genes (NBPF8, SOBP, AUTS2, RAB21, SPATA13, ABCC4, WDR7, PHLPP1, NFATC1 and MAGED1) in non-Hodgkin B cell lymphoma (B-NHL) cell line KPUM-UH1 using a high-resolution single nucleotide polymorphism array and reverse transcription polymerase chain reaction using reversely directed divergent primers within exons involved in genomic intragenic gains followed by sequencing analysis. Among them, the IERs involved in SOBP (6q21) exon 2 and 3 and AUTS2 (7q11.22) exon 2–4 were the molecular lesions specific to tumors and were frequently detected in B-NHL samples. These IERs constitute novel genetic alterations of B-NHL, which might be associated with tumorigenesis and be useful as genetic biological markers.

Several novel fusion transcripts were identified by next-generation sequencing in gastric cancer; however, the breakpoint junctions have yet to be characterized. The present study characterized a plethora of APIP-FGFR2 genomic breakpoints in the SNU-16 gastric cancer cell line, which harbored homogeneously staining regions (hsrs) and double minute chromosomes. Oligonucleotide microarrays revealed high-level amplifications at chromosomes 8q24.1 (0.8 Mb region), 10q26 (1.1 Mb) and 11p13 (1.1 Mb). These amplicons contained MYC and PVT1 at chromosome 8q24.1, BRWD2, FGFR2 and ATE1 at chromosome 10q26, and 24 genes, including APIP, CD44, RAG1 and RAG2, at chromosome 11p13. Based on these findings, reverse transcription-polymerase chain reaction (PCR) was performed using various candidate gene primers to detect possible fusion transcripts, and several products using primer sets for the APIP and FGFR2 genes were detected. Eventually, three in-frame and two out-of-frame fusion transcripts were detected. Notably, PCR analysis of the entire genomic DNA detected three distinct genomic junctions. The breakpoints were within intron 5 of APIP, which contained three distinct breakpoints, and introns 5, 7 and 9 of FGFR2. Fluorescence in situ hybridization showed several fusion signals within hsrs using two short probes (~10-kb segments of a bacterial artificial chromosome clone) containing exons 2-5 of APIP or exons 11-13 of FGFR2. Although, for any given fusion, a multiplicity of transcripts is thought to be created by alternative splicing of one rearranged allele, the results of the present study suggested that genomic fusions of APIP and FGFR2 are generated in hsrs with a diversity of breakpoints that are then faithfully transcribed.

Background Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear. Procedure This retrospective study examined the prognostic significance of CXCR4+ in pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. Results In the total cohort (n = 248), no significant differences were observed between CXCR4+ patients (n = 81) and CXCR4− patients (n = 167) in terms of 3-year overall survival (OS) (69.4% vs. 75.2%, P = 0.44). However, there was a significant difference in 3-year OS between CXCR4+ and CXCR4− patients in the low-risk (LR) group (n = 93; 79.2% vs. 98.3%, P = 0.007). CXCR4+ patients in the t(8;21) AML without KIT mutation group had a significantly worse 3-year OS than CXCR4− patients (n = 44; 76.1% vs. 100.0%, P = 0.01). Multivariate Cox regression analysis identified CXCR4+ as a poor prognostic factor for OS in LR AML patients (hazard ratio, 11.47; P = 0.01). Consistent with the data for survival analysis, CXCR4+ patients in the t(8;21) AML group had a higher incidence of splenomegaly than CXCR4− patients (25.9% vs. 5.9%, P = 0.03). Conclusions These results suggest that CXCR4+ is a poor prognostic factor for LR patients, particularly t(8;21) patients without KIT mutation. The poor outcome was only applicable to OS, not relapse-free survival (RFS); thus, CXCR4+ may be associated with a poor prognosis after recurrence. Intensive therapy, including administration of CXCR4 antagonists, may be promising for pediatric AML patients with LR.

The translocation t(8;16)(p11;p13), which results in fusion of the MOZ/MYST3/KAT6A gene (8p11) and CBP/CREBBP gene (16p13), is characterized by unique morphologic and clinical findings in acute mye...

Background Rearrangements of chromosome 8q24/MYC (8q24/MYC-r), resulting from t(8;14)(q24;q32), t(2;8)(p11;q24), or t(8;22)(q24;q11), are mainly associated with Burkitt lymphoma/leukemia (BL) and rarely observed in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The characteristics of BCP-ALL with 8q24/MYC-r are poorly understood. Procedure A retrospective nationwide study of data from patients with pediatric BCP-ALL with 8q24/MYC-r in Japan was conducted to clarify the clinical and biological characteristics associated with 8q24/MYC-r BCP-ALL. Results Ten patients with BCP-ALL with 8q24/MYC-r, including three with double-hit leukemia (DHL) (two with t(8;14)(q24;q32) and t(14;18)(q32;q21) and one with t(8;14) and t(3;22)(q27;q11)), were identified. Patients with BCP-ALL with 8q24/MYC-r had higher median age and uric acid and lactate dehydrogenase levels, than those without 8q24/MYC-r. All patients were initially treated with ALL-type chemotherapy; however, four, including one with DHL, were switched to BL-type chemotherapy, based on cytogenetic findings. One patient relapsed after standard-risk ALL-type chemotherapy, and two patients with DHL did not attain complete remission with chemotherapy; all three died within 11 months. The other seven patients treated with BL-type or high-risk ALL-type chemotherapy are alive without disease. Conclusions The clinical and laboratory features of BL with IG-MYC rearrangement, displaying a BCP immunophenotype (Wagener et al. and Herbrueggen et al. termed it as pre-BLL), are similar to those of BCP-ALL with 8q24/MYC-r. Low-risk ALL-type chemotherapy may not be appropriate for them, and further studies are required to establish an adequate therapeutic strategy. Further studies of DHL to identify new treatment strategies are also needed.

Background: Despite major improvements in survival, relapse is still a frequent and severe event in pediatric acute myeloid leukemia (AML). Cytogenetic abnormalities represent important predictors of outcome. Improved risk stratification encompassing the identification of novel cytogenetic subsets may allow refinements in treatment strategies and increase survival rates. Hypodiploidy, defined as a modal number (MN) below 46 chromosomes, is associated with an adverse outcome in pediatric acute lymphoblastic leukemia. In childhood AML, the predominant cause of hypodiploidy is loss of a sex chromosome accompanying t(8;21). The most commonly lost autosome is chromosome 7, associated with a poor prognosis. However, other chromosome losses in pediatric AML have not been independently explored. We aimed to investigate the occurrence, genetic characteristics, and prognostic impact of hypodiploidy in childhood AML. Methods: This study was conducted as a retrospective cohort study within the I-BFM-AML framework. Children 0-18 years of age diagnosed with de novo AML between January 2000 to December 2015 and a hypodiploid karyotype were eligible for inclusion. Patients with constitutional hypodiploidy, monosomy 7, composite karyotypes, and t(8;21) with loss of a sex chromosome were excluded. Cytogenetic review was performed according to International System for Human Cytogenetic Nomenclature. Hypodiploidy was considered clonal when occurring in at least three metaphases. The clone with the lowest MN defined the ploidy level. The log-rank test was applied for estimating difference in survival. Considering allografting in first complete remission (CR1) as a time-dependent event, the effect of stem cell transplantation (SCT) on overall survival (OS) was estimated by the Mantel-Byar method. Multivariate analyses included sex, complex karyotype (≥3 non-recurrent aberrations), FLT3-ITD mutation status, and white blood cell count as covariates. As all patients with FLT3-ITD mutations had same MN (MN=45) leading to a skewed imputation model, multiple imputation was not performed, and multivariate analyses included complete cases only (n=54). Results: Hypodiploidy was detected in 81/6,409 patients with full karyotyping from 14 collaborative study groups, yielding a frequency of 1.2% in childhood AML. The cohort displayed a balanced sex distribution (male/female: 42/39) and a median age of 6 (range: 0-17). MNs 45 (n=66), 44 (n=10) and 43 (n=5) were observed. No patient had MN below 43. Eight patients (9.8%) had chromosome loss as the only aberration. Forty-eight patients (59%) had a complex karyotype. Core-binding factor abnormalities were observed in three patients (3.7%). Four patients harbored FLT3-ITD mutations (9.3%, 54 tested). Median follow-time for patients alive was 4.5 years (range: 0.2-15.3). Five-year event-free survival (EFS) and OS were 34% (CI 95%: 23% - 45%) and 52% (CI 95%: 40% - 63%), respectively. Cumulative incidence of relapse was 50% with a median time-to-relapse of 0.8 years. Twelve out of 38 relapsed children (32%) were alive at the end of follow-up. Children with MN≤44 (n=15) had lower EFS (21%, CI 95%: 4% - 46%) and OS (33%, CI 95%: 10% - 59%) than children with MN=45 (n=66, EFS: 37%, CI 95%: 24% - 49%, OS: 56%, CI 95%: 42% - 68%). Crude hazard ratios (HR) for MN≤44 vs MN=45 were 1.8 (p=0.08) for EFS and 1.9 (p=0.1) for OS. After adjustment HRs increased to 4.9 (p=0.001) and 6.1 (p=0.003), respectively. Loss of chromosome Y (n=12) displayed a non-significant superior survival (EFS: 58%, OS: 75%). Poor survival rates were especially observed in monosomy 9 (n=11, EFS: 14%, OS 15%), 10 (n=5, EFS: 20%, OS: 20%), and 16 (n=5, EFS: 25%, OS: 25%), although not reaching statistical significance. Allogenic SCT did not offer any advantage in survival. For children receiving SCT in CR1 (n=18), crude HR for OS was 1.4, p=0.39. Multivariate analysis showed HR for OS at 1.5, p=0.42. Exclusion of children not reaching CR1 (n=7) did not significantly change HRs. This large international study reveals that hypodiploidy is rare in pediatric AML. Children with loss of chromosome 9, 10, or 16 all displayed unfavorable survival rates, whereas loss of chromosome Y did not have influence on prognosis. SCT in CR1 did not improve survival. Our results suggest that modal number has an impact on outcome and may be important for risk stratification. Disclosures No relevant conflicts of interest to declare.

Background Recent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, approximately 40% of patients with pediatric AML relapse, contributing to a relatively low overall survival (OS) rate of approximately 70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. Methods We performed transcriptome analysis (RNA-seq) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial and investigated correlations between genetic aberrations and clinical information. Findings Using RNA-seq, we identified 54 in-frame gene fusions and one RUNX1 out-of-frame fusion in 53 out of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through RT-PCR and RNA-seq. Six gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, NPM1-DNAJC1, TBL1XR1-RARB, ETV6-CTNNB1, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. Interpretation RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially driver alterations in nearly all patients with AML, including novel gene fusions. Based on the results of the RNA-seq, risk stratification should be reconsidered. Funding Statement: Ministry of Education, Culture, Sports, Science, and Technology of Japan and Japan Agency for Medical Research and Development. Declaration of Interests: I have no financial relationships to disclose. Ethics Approval Statement: The present study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards of Gunma Children’s Medical Center and the participating institutes and the ethical review board of the JPLSG AML-05 trial.

Background On the basis of results of previous Japanese trials for myeloid leukemia in Down syndrome (ML-DS), the efficacy of risk-oriented therapy was evaluated in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D05 study. Procedure All patients received induction chemotherapy that consisted of pirarubicin, intermediate-dose cytarabine, and etoposide. Patients who achieved complete remission (CR) after initial induction therapy were stratified to the standard risk (SR) group and received four courses of reduced-dose intensification therapy. Patients who did not achieve CR were stratified to the high risk (HR) group and received intensified therapy that consisted of continuous or high-dose cytarabine. Results A total of 72 patients were eligible and evaluated. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and two patients to HR. No therapy-related deaths were observed during intensification therapy. The 3-year event-free and overall survival rates were 83.3% ± 4.4% and 87.5% ± 3.9%, respectively. Age at diagnosis less than 2 years was a significant favorable prognostic factor for risk of relapse (P = 0.009). Conclusions The attempt of risk-oriented prospective study for ML-DS was unsuccessful, but despite the dose reduction of chemotherapeutic agents, the overall outcome was good, and further dose reduction might be possible for specific subgroups.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin-1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma-derived cell lines (HMCLs) expressed non-translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co-expressed wild type transcripts (wt.DCC), while eight co-expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient-derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

Myeloid malignancy with Down syndrome (ML-DS) is estimated to have a step-wise leukemogenesis including GATA1 mutation. Trisomy 21 is essential for ML-DS; however, we do not know exactly which gene or genes located on chromosome 21 are necessary for the ML-DS. We report a female infant with transient myeloproliferative disorder (TMD) and partial trisomy 21. SNP array analysis showed 10 Mb amplification of 21q22.12-21q22.3, which included DYRK1A, ERG, and ETS but not the RUNX1 gene. With two other reported TMD cases having partial trisomy 21, DYRK1A, ERG, and ETS were the most likely genes involved in collaboration with the GATA1 mutation.

We have identified a novel SPAG9-JAK2 fusion in a B-cell precursor acute lymphoblastic leukemia (ALL) with t(9;17)(p24;q21) and a poor outcome, using paired-end transcriptome sequencing. Homozygous and hemizygous deletions of CDKN2A/2B, and hemizygous deletions of PAX5, BTG1, CDK6, ADARB2, and IKZF1 were also identified by multiple ligation-dependent probe amplification and single nucleotide polymorphism array analyses. Having both a tyrosine kinase-activating rearrangement and genomic lesions affecting lymphoid transcription factors suggested that the leukemia was of the Philadelphia chromosome (Ph)/BCR-ABL1-like ALL subtype and that JAK2 inhibitors might be able to overcome this aggressive ALL with SPAG9-JAK2.

Acute lymphoblastic leukemia (ALL) occasionally develops before or after the onset of Langerhans cell histiocytosis (LCH). The mechanism of LCH developing after ALL remains unclear; thus the clonality of LCH developing during maintenance chemotherapy for T-cell ALL (T-ALL) was investigated. The T-ALL and LCH cells tested had the same T-cell receptor (TCR) gamma rearrangement. Mutation analysis of the NOTCH1 gene revealed 7213C>T (Q2405X) in exon 34 in T-ALL and LCH cells, but 5156T>C (I1719T) in exon 27 only in T-ALL. Polymerase chain reaction-restriction fragment length polymorphism analysis revealed three patterns of NOTCH1 mutations in T-ALL cells. The results suggest that the T-ALL and LCH cells were derived from a common precursor with TCR rearrangement and a single NOTCH1 mutation, rather than LCH cells developing from a minor clone of T-ALL with single NOTCH1 mutation.

Investigation of genetic alterations associated with relapse in acute lymphoblastic leukemia (ALL) may help to identify druggable targets for specific therapies. Early T-cell precursor ALL (ETP-ALL) is a subtype of T-ALL with poor prognosis. Although the genetic landscape of ETP-ALL has been determined, genetic alterations related to the relapse of ETP-ALL have not been fully investigated. Here, we report the first patient with relapsed pediatric ETP-ALL to exhibit a homozygous JAK3 activating mutation, V674A, caused by acquired uniparental disomy (UPD). Single nucleotide polymorphism array analysis revealed acquired UPD (aUPD) at the 19p13.3-p12 locus only in leukemic cells at relapse. Sanger sequence of the JAK3 gene, which was located at 19p13.1 and frequently mutated in ETP-ALL, was performed in paired leukemic samples to determine homozygous JAK3 V674A mutation only in relapsed leukemic cells. In contrast, leukemic cells at initial diagnosis harbored hemizygous JAK3 V674A mutation. Further, whole-exome sequencing revealed mutations in 18 genes only in relapsed samples, although none of these was recurrent in T-ALL. These findings suggest that aUPD at 19p13.1 is partly associated with relapse in this patient. Pharmacological inhibition of JAK3 may be therapeutic in such cases.

[Background] Customized gene panel sequencing is commonly used in cancer research to detect point mutations and small insertions or deletions. This technique can also be used to detect copy number alterations (CNAs) in tumor samples based on the comparison of sequence depth of targeted regions between samples and variant allele frequencies of germline single nucleotide polymorphisms (SNPs). CNAs in TP53 and CDKN2A/2B are frequently found in various types of leukemias. TP53 loss was reported to be a frequent CNA with poor prognosis in adult AML, and CDKN2A/2B alterations were found to be frequent in adult and pediatric acute lymphoblastic leukemia. However, the molecular and clinical profiles of the CNAs of these genes remain unclear in pediatric AML. [Patients and Methods] We analyzed clinical samples from 331 pediatric AML patients in the AML-05 trial, which was conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group. Targeted sequencing was performed using a 343-gene custom panel and next-generation sequencer. These 343 genes are reportedly associated with hematopoietic malignancy or solid tumor pathogenesis. Copy number analysis was performed using the in-house pipeline CNACS. Additionally, a SNP array was performed for 40 of the 331 patients, including 34 patients with complex karyotype, to detect CNAs of TP53. [Results] TP53 alterations were identified in 7 (2.1%) of 331 patients. TP53 losses and mutations were detected in 7 and 4 patients, respectively, and all the patients with TP53 mutations concurrently had TP53 losses. Seven patients with TP53 losses were identified by CNACS, although only 4 patients with TP53 losses were found by SNP array. As for the cytogenetics of 7 patients with TP53 losses, six carried a complex karyotype, lacking FAB-M7 morphology. Accordingly, 6 (28.6%) of 21 non-FAB-M7 patients with complex karyotype had TP53 losses. Fusion genes and gene mutations were rare in patients with TP53 losses (NUP98-KDM5A, n=1; KIT, n=1; NRAS, n=1; CEBPA, n=1; ASXL1, n=1), and no patients with core-binding factor AML (CBF-AML) or KMT2A-rearrangements had TP53 losses. In survival analyses, patients with TP53 losses had significantly worse overall survival, event-free survival, and cumulative incidence of relapse rates than patients with wildtype TP53 (14.3% vs 71.4%, p CDKN2A/2B alterations were identified in 10 patients (3.0%) (9 losses and 1 mutation). Three patients with CDKN2A/2B losses had simultaneous TP53 alterations. Unlike TP53 alterations, CDKN2A/2B alterations were found in patients with CBF-AML and those with FAB-M7. In survival analyses, no significant difference was observed between patients with or without CDKN2A/2B alterations. [Discussion] TP53 losses were identified in approximately 2% of patients with pediatric AML. However, these alterations were found to be frequent in patients with complex karyotypes, particularly in those with non-FAB-M7 (28.6%). Furthermore, patients with TP53 losses had very poor prognosis. These findings indicate the necessity of risk stratification using TP53 status for pediatric AML. The efficacy of hematopoietic stem cell transplantation (HSCT) for the treatment of patients with TP53 losses remains unclear because 6 of 7 patients with TP53 losses received HSCT in this study. Moreover, because some of the identified TP53 mutations were possible germline mutations, HSCT may increase the incidence rate of the secondary cancer in patients with TP53 mutations. Thus, the efficacy of new agents (e.g., azacitizine and gemtuzumab ozogamicin) should be evaluated in future clinical trials for the intensification of chemotherapy for these patients. Meanwhile, the clinical and molecular profiles of CDKN2A/2B alterations were not fully identified in this study. However, the high incidence of CDKN2A/2B alterations in patients with TP53 losses indicates that the correlation between these gene alterations is essential for oncogenesis in a subset of pediatric AML patients. Figure Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

Background: Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy thus results in a favorable outcome. However, relapsed and refractory cases are rarely salvageable, regardless of receiving hematopoietic stem cell transplantation. Several factors such as certain chromosomal abnormalities and age at diagnosis are somewhat prognostic, but no universal prognostic factor has been found to date. In order to identify a subgroup with high risk of treatment failure, the role of minimal residual disease (MRD) with three methods were explored in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) trial AML-D11. Procedure: AML-D11 is a nationwide single-arm clinical trial for children (4 months to 17 years old) with ML-DS. All patients received an identical chemotherapy to the previous AML-D05 study (Taga T. Pediatr Blood Cancer 2016). MRD was evaluated at two time points, one after the induction therapy and another at the end of whole chemotherapy, using 3 different methods; flow cytometric MRD (FCM-MRD), deep sequencing MRD of mutant GATA1 (GATA1-MRD) and PCR MRD of WT1 mRNA expression (WT1-MRD). WT1-MRD was measured in both bone marrow (BM) and peripheral blood (PB) samples, while FCM- and GATA1-MRD were measured only in BM samples. Results: A total of 78 patients were eligible and followed-up with a median of 47.6 months (range, 8 to 68.8 months). Seventy-six patients were stratified to the standard risk (SR) and one patient to the high risk (HR) group by morphological response. One patient died of sepsis during initial induction therapy. Three-year event-free survival (EFS) and overall survival (OS) rates were 87.2% (95%CI, 77.5 to 92.9%) and 89.7% (95%CI, 80.5 to 94.7%), respectively. FCM-MRD and GATA1-MRD after initial induction therapy were positive in 5/65 and 7/59 patients, respectively, which were both significantly prognostic (Fig.1). Prognostic significance of WT1-MRD could not be evaluated due to a limited number of collected samples. Conclusions: MRD detections by FCM and targeted deep sequencing of GATA1 after initial induction therapy are both significant prognostic factors for predicting relapse. Risk stratification using FCM-MRD is currently incorporated in the on-going Japan Children's Cancer Group ML-DS trial (AML-D16; jrct.niph.go.jp, jRCTs041190047). Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction Treatment outcomes of pediatric acute myeloid leukemia (AML) have improved via stratification therapy. However, relapse and mortality are still observed in 40% and 30% patients, respectively. Recently, comprehensive analyses using next-generation sequencing have revealed novel genetic alterations in adult AML patients, but insufficient analyses have been performed in pediatric AML patients. Further studies are warranted to evaluate the relationship between a more detailed genetic background and prognosis in pediatric AML patients. Methods We performed targeted sequencing using a 343-gene custom panel and next-generation sequencer in 302 pediatric patients with de novo AML who participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group from 2006 to 2010. One hundred and twenty patients with core binding factor (CBF)-AML, 52 with KMT2A rearrangements, 56 with normal karyotype (NK), 18 with complex karyotype (CK), and 57 with other karyotypes except for patients with acute megakaryocytic leukemia were enrolled. Correlations among gene mutations, other cytogenetic alterations, and clinical characteristics were investigated. Results and Discussion We found the following 6 recurrent gene mutations, which have not been frequently reported in pediatric AML: KMT2C (n = 13, 4.3%), PHF6 (n = 6, 2.0%), MGA (n = 13, 4.3%), TET2 (n = 11, 3.6%), JAK3 (n = 12, 4.0%), and GATA2 (n = 11, 3.6%). KMT2C mutations have been reported in adult and pediatric patients with CBF-AML and in a few patients with other types of AML; however, their clinical significance has not been revealed. Six of 13 patients with KMT2C mutations were found in CBF-AML, whereas the other 7 patients were detected in non CBF-AML (1 patient in KMT2A-rearrangement, 1 in CK, and 5 in other karyotypes). Loss of function mutations in KMT2C have also been reported to be associated with chemotherapy resistance in pediatric AML patients. Although no significant differences were found, 4 of 13 patients with KMT2C mutations did not achieve complete remission (p = .072), and 3 patients relapsed in In adult AML, PHF6 mutations have been detected in 3% of patients and have been reported as poor prognostic factors. In 6 patients with PHF6 mutations, 3 patients had a normal karyotype, and 2 patients had a complex karyotype. No PHF6 mutations were detected in AML patients with CBF or KMT2A rearrangements. PHF6 mutations were significantly frequently detected in AML patients with myelodysplasia-related changes (4/6, p = .008). Although 5 of 6 patients with PHF6 mutations archived complete remission, finally, 5 patients relapsed or died. There were significant differences in EFS between patients with or without PHF6 mutations (3-year EFS 16.7% vs. 58.2%; p = .041). MGA mutations have been reported thus far in a few AML patients. MGA is a negative regulator of MYC signaling; thus, loss of functions of MGA is considered to contribute to tumorigenesis. However, the clinical significance of MGA mutations has not been reported. MGA mutations were significantly frequently detected in AML patients with FAB M2 (7/13, p = .020). In our cohort, 2 of 3 AML patients with DEK-NUP214 had MGA mutations (p = .004). We did not find correlations between MGA mutations and prognosis (3-year OS 83.3% vs. 75.5%; p= .488, 3-year EFS 66.7% vs. 56.7%; p = .507) TET2 mutations have been found in 8%-19% of adult AML cases and are considered poor prognostic factors. In previous reports, TET2 mutations were also detected in 3.8% of pediatric AML patients, but because of the small number of patients, the association between TET2 mutations and prognosis has not been revealed. TET2 mutations were significantly frequently detected in older AML patients (median age 13.95 vs. 9.0 years; p = .022) and in AML patients with NK (5/11, p = .035). Contrary to previous reports on adult AML, TET2 mutations were not associated with prognosis (3-year OS 77.9% vs. 75.9%; p= .688, 3-year EFS 54.5% vs. 57.4%; p = .907). This suggested that TET2 mutations have a different clinical significance between adult and pediatric AML patients. In conclusion, mutations in the KMT2C and PHF6 genes were associated with poor prognosis, but TET2 mutations were not. Further analysis should be needed about TET2 mutations. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

BACKGROUND: Outcome of infants with acute lymphoblastic leukemia (ALL), especially those with rearrangement of MLL (KMT2A) gene (MLL-r), is extremely poor. A strategy to perform allogeneic hematopoietic stem cell transplantation (HSCT) for all the infants with MLL-r ALL in first remission (1CR) have been tested in the previous Japanese trials MLL96/98/03, however, the improvement was modest. Given the recent evidence of a limited role of HSCT especially in infants lacking poor prognostic factors, efficacy and safety of an intensive chemotherapy and risk stratification to limit HSCT for only infants with high-risk of relapse were evaluated in the JPLSG MLL-10 trial (UMIN000004801). PATIENTS & METHODS: Infants age less than 365 days with ALL were registered in the MLL-10 study and were stratified by their MLL gene status, age at diagnosis, and presence of CNS disease; low-risk (LR), if the patients had germline MLL gene (MLL-g); intermediate-risk (IR), if the patients with MLL-r ALL were age 180 days or older and lack CNS disease; high-risk (HR), if the patients with MLL-r ALL were age RESULTS: A total of 90 eligible infants with ALL were registered in the MLL-10 study between Jan/2011 and Dec/2014; 15 cases were stratified as LR, 19 as IR, and 56 as HR. Remission status was evaluated after 2 chemotherapy courses; 82 (91.1%) achieved 1CR, 3 failed to achieve 1CR, and 5 discontinued the trial before CR evaluation. No early death was observed. With median follow-up period of 1954 days (range, 534-2835 days) in the live patients, 3-year probability of event-free survival (pEFS) and overall survival (pOS) were 70.9% (95% CI, 60.0-79.3%) and 86.6% (77.6-92.2%), respectively. Among the MLL-r cases, 3-year pEFS and pOS were 66.2% (53.9-75.9%) and 83.9% (73.4-90.5%), respectively. According to the risk groups, 3-year pEFS were 93.3% (61.2-99.0%) for LR, 94.4% (66.6-99.2%) for IR, and 56.6% (42.4-68.6%) for HR cases. Regarding the MRD studies, correlation of MRD results in 3 methodologies seemed reasonable, but while flow-MRD could be evaluated in 85 cases, MLL-fusion PCR-MRD (MLL-r cases only) and IgH/TCR PCR-MRD could only be evaluated in 55 and 50 cases, respectively. In the univariable analysis for MLL-r cases, female sex (P=0.04), younger age at diagnosis (P=0.01), and 0.01%< flow-MRD after 2 courses of chemotherapy (P CONCLUSIONS: Introduction of intensive chemotherapy enabled us to spare allogeneic HSCT in 1CR at least in a subset of infants with MLL-r ALL without compromising their outcome. This was accomplished also because of the aggressive supportive care provided to the study cases, such as full hospitalization during and after the intensive treatment phases, intensive use of rasburicase to prevent tumor lysis syndromes, and aggressive infection prophylaxis. However, outcome of HR MLL-r cases is still unsatisfactory, and introduction of novel agents is mandatory for further improvement in the outcome of infants with ALL. Figure Disclosures No relevant conflicts of interest to declare.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear.This retrospective study examined the prognostic significance of CXCR4+ in pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study.In the total cohort (n = 248), no significant differences were observed between CXCR4+ patients (n = 81) and CXCR4- patients (n = 167) in terms of 3-year overall survival (OS) (69.4% vs. 75.2%, P = 0.44). However, there was a significant difference in 3-year OS between CXCR4+ and CXCR4- patients in the low-risk (LR) group (n = 93; 79.2% vs. 98.3%, P = 0.007). CXCR4+ patients in the t(8;21) AML without KIT mutation group had a significantly worse 3-year OS than CXCR4- patients (n = 44; 76.1% vs. 100.0%, P = 0.01). Multivariate Cox regression analysis identified CXCR4+ as a poor prognostic factor for OS in LR AML patients (hazard ratio, 11.47; P = 0.01). Consistent with the data for survival analysis, CXCR4+ patients in the t(8;21) AML group had a higher incidence of splenomegaly than CXCR4- patients (25.9% vs. 5.9%, P = 0.03).These results suggest that CXCR4+ is a poor prognostic factor for LR patients, particularly t(8;21) patients without KIT mutation. The poor outcome was only applicable to OS, not relapse-free survival (RFS); thus, CXCR4+ may be associated with a poor prognosis after recurrence. Intensive therapy, including administration of CXCR4 antagonists, may be promising for pediatric AML patients with LR.