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Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domain was invariably affected. Our results highlight the role of RNase IIIb domain mutations in DICER1 along with TP53 inactivation in PPB pathogenesis. Cancer Res; 74(10); 2742–9. ©2014 AACR.

PDF file - 1805KB, Supplementary Table S1. List of primers used for validation of somatic mutations detected by whole-exome sequencing. Supplementary Table S2. Primers for DICER1 sequencing of all coding exons. Supplementary Table S3. Primers for DICER1 sequencing of all coding exons in paraffin embedded samples. Supplementary Table S4. Primers for TP53, UBA2, PDCD2L, CTNNB1, and GPR182. Supplementary Table S5. List of mutated genes detected by whole-exome sequencing. Supplementary Table S6. Allele frequency of DICER1 mutations in cases with unknown somatic status. Supplementary Table S7. Recurrent mutations detected by whole-exome sequencing. Supplementary Figure S1. Analyzing processes using Genomon. Supplementary Figure S2. Mean coverage of whole-exome sequencing in primary tumor, relapse tumor, and control samples. Supplementary Figure S3. Mean coverage of whole exome sequencing with GC contents in primary tumor, relapse tumor and control samples. Supplementary Figure S4. Detected mutations of DICER1 by Sanger sequencing. Supplementary Figure S5. Distribution of genetic lesions with 14 samples in PPB cases. Supplementary Figure S6. Common amplification lesion at 19q13.11.

Transient abnormal myelopoiesis (TAM) is a unique clonal myeloproliferation characterized by immature megakaryoblasts that occurs in 5–10% of neonates with Down syndrome (DS). Although TAM regresses spontaneously in most patients, approximately 20% of TAM cases result in early death, and approximately 20% of survivors develop acute megakaryoblastic leukemia (AMKL). We retrospectively reviewed records of 35 DS patients with TAM to determine the correlation between clinical characteristics and blast percentage. Thirteen of the 35 patients were classified as low blast percentage TAM (LBP-TAM), defined as TAM with a peak peripheral blast percentage ≤ 10%. Although no patient with LBP-TAM experienced systemic edema, disseminated intravascular coagulation, or early death, eight patients had elevated direct bilirubin levels (> 2 mg/dl) and one developed AMKL. All patients with LBP-TAM had serum markers of liver fibrosis that exceeded the normal limits, and two patients underwent liver biopsy to clarify the etiology of pathological jaundice. Taken together, our results suggest that patients with LBP-TAM may be at risk of liver fibrosis and liver failure, similarly to patients with classical TAM. Although these patients generally have a good prognosis, they should be carefully monitored for potential development of liver disease and leukemia.

Human herpesvirus-6 (HHV-6) is a common pathogen affecting the human population. Primary HHV-6 infection generally occurs during infancy and causes exanthema subitum. Moreover, HHV-6 may exhibit inherited chromosomally integrated HHV-6 (iciHHV-6) in certain individuals. Although iciHHV-6 is generally known to be nonpathogenic, it may cause reactivation in patients with primary immunodeficiency disease (PID). XIAP deficiency is a rare PID characterized by recurrent hemophagocytic lymphohistiocytosis (HLH). It has been reported that the Epstein-Barr virus primarily causes HLH; however, the other pathogens, including HHV-6, can also cause this complication. We encountered a case of XIAP deficiency accompanied by iciHHV-6. He suffered from recurrent HLH, for which allogeneic bone marrow transplantation (BMT) was performed as a curative therapy. During the course of BMT, the patient experienced HLH three times, but there was no reactivation of endogenous HHV-6 from iciHHV-6. Finally, the patient achieved complete donor chimerism and a decline in HHV-6 DNA copy number in whole blood. This case report demonstrates no evidence of reactivation of iciHHV-6 during BMT in a patient with XIAP deficiency.

Fusion genes involving MEF2D have recently been identified in precursor B-cell acute lymphoblastic leukemia, mutually exclusive of the common risk stratifying genetic abnormalities, although their true incidence and associated clinical characteristics remain unknown. We identified 16 cases of acute lymphoblastic leukemia and 1 of lymphoma harboring MEF2D fusions, including MEF2D-BCL9 (n=10), MEF2D-HNRNPUL1 (n=6), and one novel MEF2D-HNRNPH1 fusion. The incidence of MEF2D fusions overall was 2.4% among consecutive precursor B-cell acute lymphoblastic leukemia patients enrolled onto a single clinical trial. They frequently showed a cytoplasmic μ chain-positive pre-B immunophenotype, and often expressed an aberrant CD5 antigen. Besides up- and down-regulation of HDAC9 and MEF2C, elevated GATA3 expression was also a characteristic feature of MEF2D fusion-positive patients. Mutations of PHF6, recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. MEF2D fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treatment, MEF2D fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of CDKN2A/CDKN2B gene deletions. Our observations indicate that MEF2D fusions comprise a distinct subgroup of precursor B-cell acute lymphoblastic leukemia with a characteristic immunophenotype and gene expression signature, associated with distinct clinical features.

TO THE EDITOR: Acute myeloid leukemia (AML) is a complicated disease characterized by the uncontrolled proliferation of hematopoietic precursors and the loss of differentiation ability caused by various genetic alterations. Recent advances in massively parallel sequencing technologies have

Introduction: Transient abnormal myelopoiesis (TAM), also known as transient leukemia or transient myeloproliferative disorder, is a unique clonal myeloproliferation characterized by immature megakaryoblasts occurring in 10% of neonates with Down syndrome. Although most patients show spontaneously resolution of TAM without therapeutic interventions, approximately 20% of TAM cases result in early deaths, i.e., within 9 months, and approximately 20% of the survivors develop acute megakaryoblastic leukemia (AMKL) within 4 years. A somatic GATA1 gene mutation that leads to the exclusive expression of a truncated GATA1 protein is shared by both TAM and AMKL cells. According to previous reports, cytokine levels are associated with liver failure, which is a cause of early death. Here, we analyzed 154 DS patients with TAM enrolled in the TAM-10 prospective observational study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group to determine the association between clinical characteristics and cytokine levels in such patients. Patients and Methods: A total of 167 neonates (89 boys and 78 girls) diagnosed with TAM were prospectively registered in the TAM-10 study between May 2011 and February 2014. We analyzed cytokine levels in 154 of the 167 enrolled patients whose samples were available. Somatic GATA1 gene mutations were confirmed in 151 (98%) of 154 patients using Sanger and/or next-generation sequencing. Using the Bio-Prex cytokine assay, serum concentrations of the following 27 cytokines were measured: interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins alpha and beta (MIP-1α and MIP-1β), eotaxin, interferon gamma-induced protein (IP-10), regulated upon activation, normal T-cell expressed and secreted, interleukin I receptor agonist (IL-RA), and 13 different interleukins (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, and IL-17). For all analyses, P values were two-tailed and a P value of Results: The median (range) white blood cell (WBC) count at diagnosis was 37.2 (2.4-478.7) × 10 9 cells/L. Forty-seven (31%) of 154 patients received low-dose cytarabine. When we compared 29 patients with a high WBC count (≥100 × 10 9 cells/L, known as a poor prognostic factor in TAM patients) to 125 patients without a high WBC count for 27 cytokine levels, the levels of 16 cytokines (IL-1b, IL-1ra, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, Eotaxin, PDGF-bb, basic FGF, G-CSF, GM-CSF, MCP-1b, and VEGF) were significantly higher in patients with a high WBC group. Early death occurred in 14 (9%) of 154 patients. Cytokine levels were compared between the early death group (n = 14) and remaining patients (n = 140) and it was observed that the levels of 10 cytokines (IL-1b [p = 0.016], IL-1ra [p < 0.001], IL-6 [p < 0.001], IL-7 [p = 0.009], IL-8 [p < 0.001], IL-10 [p = 0.014], IL-13 [p = 0.002], MCP-1 [p = 0.030], MIP-1b [p = 0.024], and TNF-α [p = 0.008]) were significantly higher in the early death group. When the patients were divided in two groups according to the median IL-1β concentration showing the lowest p-value, the early death rate in the IL-1β high group was significantly higher than that in the IL-1β low group (16% vs. 3%, p < 0.001). IL-1, IL-6, IL-8, and TNF-α are proinflammatory cytokines induced by MCP-1 and MIP1-b. Early death was strongly associated with hypercytokinemia, suggesting that therapeutic interventions (e.g., systemic steroid therapy) may be effective in patients with hypercytokinemia. However, there was no relation between the levels of 27 cytokines and leukemia development. Conclusion: Our findings suggested that measurement of cytokine levels may be a useful marker for predicting early death and an indicator of therapeutic interventions required in TAM patients. Disclosures No relevant conflicts of interest to declare.

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.

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.

Recent reports described the NUP98-NSD1 fusion as an adverse prognostic marker for acute myeloid leukaemia (AML) and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harbouring NUP98-NSD1 fusion. PRDM16 gene expression levels were measured via real-time polymerase chain reaction in 369 paediatric patients with de novo AML, of whom 84 (23%) exhibited PRDM16 overexpression (PRDM16/ABL1 ratio ≥0·010). The frequencies of patients with high or low PRDM16 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 4% vs. 96%, P

High PRDM16 (also known as MEL1) expression is a representative marker of acute myeloid leukemia (AML) with NUP98-NSD1 and is a significant predictive marker for poor prognosis in pediatric AML. However, the clinical features of adult AML with PRDM16 expression remain unclear. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of MECOM (also known as EVI1). We investigated PRDM16 expression in 151 AML patients, with 47 (31%) exhibiting high PRDM16 expression (PRDM16/ABL1 ratio ≥ 0.010). High PRDM16 expression significantly correlated with DNMT3A (43% vs. 15%, P

Background Kasabach-Merritt phenomenon (KMP) is a rare condition and optimal treatments have not yet been established, especially for cases that are unresponsive to first-line therapy. We retrospectively reviewed 11 KMP cases treated over the past 13 years in our institute. Observations With the exception of 1 case, steroids were administered as the first-line therapy. Eight cases required second-line or third-line therapy. The effective salvage therapies include interferon (n=1), radiotherapy (n=1), and chemotherapy (n=5). One case continues to depend upon chemotherapy. Three refractory cases were therapy dependent over 1 year of age, whereas 8 were treated effectively by 6 months of age. Conclusions Chemotherapy seems to be the most effective therapy for steroid-resistant KMP cases.

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome-related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

ASXL2 is an epigenetic regulator involved in polycomb repressive complex regulation or recruitment. Clinical features of pediatric acute myeloid leukemia (AML) patients with ASXL2 mutations remain unclear. Thus, we investigated frequencies of ASXL1 and ASXL2 mutations, clinical features of patients with these mutations, correlations of these mutations with other genetic alterations including BCOR/BCORL1 and cohesin complex component genes, and prognostic impact of these mutations in 369 pediatric patients with de novo AML (0-17 years). We identified 9 (2.4%) ASXL1 and 17 (4.6%) ASXL2 mutations in 25 patients. These mutations were more common in patients with t(8;21)(q22;q22)/RUNX1-RUNX1T1 (ASXL1, 6/9, 67%, P = 0.02; ASXL2, 10/17, 59%, P = 0.01). Among these 25 patients, 4 (27%) of 15 patients with t(8;21) and 6 (60%) of 10 patients without t(8;21) relapsed. However, most patients with relapse were rescued using stem cell transplantation irrespective of t(8;21). The overall survival (OS) and event-free survival (EFS) rates showed no differences among pediatric AML patients with t(8;21) and ASXL1 or ASXL2 mutations and ASXL wild-type (5-year OS, 75% vs. 100% vs. 91% and 5-year EFS, 67% vs. 80% vs. 67%). In 106 patients with t(8;21) AML, the coexistence of mutations in tyrosine kinase pathways and chromatin modifiers and/or cohesin complex component genes had no effect on prognosis. These results suggest that ASXL1 and ASXL2 mutations play key roles as cooperating mutations that induce leukemogenesis, particularly in pediatric AML patients with t(8;21), and these mutations might be associated with a better prognosis than that reported previously.

Mutations in RAS are frequent in acute myeloid leukemia (AML), and are thought to contribute to leukemogenesis in a subset of patients; however, their prognostic significance has not been firmly established. One hundred and fifty-seven pediatric patients with AML were analyzed for NRAS and KRAS mutations around hot spots at codons 12, 13, and 61. Twenty-nine patients (18.5%) had an activating mutation of RAS. We found KRAS mutations to be more frequent than NRAS mutations (18/29, 62.1% of patients with RAS mutation), in contrast to previous reports (18-40%). The frequency of RAS mutation was higher in French-American-British types M4 and M5 than other types (P = 0.02). There were no significant differences in other clinical manifestations or distribution in cytogenetic subgroups, or aberrations of other genes, including KIT mutation, FLT3-ITD, and MLL-PTD, between patients with and without RAS mutations. No significant differences were observed in the 3-year overall survival and disease-free survival; however, the presence of RAS mutation was related to late relapse. The occurrence of clinical events at relatively late period should be monitored for in AML patients with mutations in RAS.

A 6-year-old Japanese female was diagnosed as having myeloid/NK cell precursor acute leukemia (MNKL) using immunocytochemical analysis. The patient was treated by cord blood transplantation from an HLA 1-locus mismatched unrelated donor after chemotherapy comprising cytosine arabinoside, idarubicin, etoposide, and L-asparaginase. We detected a nonsense mutation, C7412A, resulting in S2471X, where X is a terminal codon, in the PEST domain of NOTCH1 in this patient. The presence of the NOTCH1 activating mutation in MNKL might suggest a possible role in the leukemogenesis of MNKL. Pediatr Blood Cancer. 2010;55:1406–1409. © 2010 Wiley-Liss, Inc.

Background Diamond-Blackfan anemia is a rare, clinically heterogeneous, congenital red cell aplasia: 40% of patients have congenital abnormalities. Recent studies have shown that in western countries, the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes in about 50% of patients. There have been no studies to determine the incidence of these mutations in Asian patients with Diamond-Blackfan anemia. Design and Methods We screened 49 Japanese patients with Diamond-Blackfan anemia (45 probands) for mutations in the six known genes associated with Diamond-Blackfan anemia: RPS19 , RPS24 , RPS17 , RPL5 , RPL11 , and RPL35A. RPS14 was also examined due to its implied involvement in 5q- syndrome. Results Mutations in RPS19 , RPL5 , RPL11 and RPS17 were identified in five, four, two and one of the probands, respectively. In total, 12 (27%) of the Japanese Diamond-Blackfan anemia patients had mutations in ribosomal protein genes. No mutations were detected in RPS14 , RPS24 or RPL35A . All patients with RPS19 and RPL5 mutations had physical abnormalities. Remarkably, cleft palate was seen in two patients with RPL5 mutations, and thumb anomalies were seen in six patients with an RPS19 or RPL5 mutation. In contrast, a small-for-date phenotype was seen in five patients without an RPL5 mutation. Conclusions We observed a slightly lower frequency of mutations in the ribosomal protein genes in patients with Diamond-Blackfan anemia compared to the frequency reported in western countries. Genotype-phenotype data suggest an association between anomalies and RPS19 mutations, and a negative association between small-for-date phenotype and RPL5 mutations.

We report a rare case of neonatal Beckwith-Wiedemann syndrome (BWS) associated with alveolar rhabdomyosarcoma (RMS). Alveolar RMS was diagnosed on the basis of excisional biopsy. Chemotherapy was initiated and followed by bone marrow transplantation. The patient, who is now 3 years and 11 months of age, is alive 46 months after the initial diagnosis, albeit with disease. We could not detect the PAX3-FKHR or PAX7-FKHR transcripts; however, we could observe hypomethylation of the differentially methylated region of the long QT intronic transcript 1. Thus, neonatal alveolar RMS with BWS may result from an alternate molecular pathway.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. The simultaneous loss of Ink4a/Arf function and disruption of Met signaling in Ink4a/Arf-/- mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF) induces RMS with extremely high penetrance and short latency. To address the roles of MET and CDKN2A (p16INK4A/p14ARF) in human RMS, we performed mutational analyses in 39 samples of RMS by PCR-SSCP. No mutations were detected in exons 14-21 of MET whereas a nonsense mutation at codon 80 of p16(INK4A) was identified in an alveolar RMS cell line. We also quantified the relative expression levels and DNA copy numbers of these genes in seven cell lines and 17 fresh tumors by real-time quantitative PCR. Expression of MET was detected in all samples; however, more than 10-fold difference was found in the samples with higher or lower expression level, despite a normal DNA copy number. The protein expression level was consistent with that of mRNA, and in cell lines with a higher expression level, MET was constitutively activated. Notably, the expression level of MET was significantly higher in patients who died (P = 0.02), in patients with stage IV (P = 0.04), as well as in patients with PAX3-FKHR chimeric transcript (P = 0.04). On the other hand, reduced or absent expression of p16INK4A and/or p14(ARF) showed no significant correlation with the clinicopathological parameters, except for the age at diagnosis. Our data suggest that MET plays a role in the progression of RMS.

Imatinib, the ABL kinase inhibitor, is used not only for Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia, but also for Ph + acute lymphoblastic leukemia (ALL), although resistance to the drug tends to develop in an early stage of the clinical course. We describe a childhood refractory Ph + ALL patient in whom progressive resistance to imatinib was correlated with the appearance of a mutation in the BCR-ABL kinase domain and in vitro drug resistance to imatinib as determined by the methyl-thiazol-tetrazolium (MTT) assay. A missense mutation of T to C (Y253H) of the ABL gene was identified in the resistant clone, suggesting that this mutation may play an etiological role in the rapid loss of drug sensitivity.

Familial platelet disorder with a propensity to develop acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disease characterized by thrombocytopenia, abnormal platelet function, and a propensity to develop myelodysplastic syndrome (MDS) and AML. So far, > 20 affected families have been reported. Recently, a second RUNX1 alteration has been reported; however, no additional molecular abnormalities have been found so far. We identified an acquired CBL mutation and 11q-acquired uniparental disomy (11q-aUPD) in a patient with chronic myelomonocytic leukemia (CMML) secondary to FPD with RUNX1 mutation but not in the same patient during refractory cytopenia. This finding suggests that alterations of the CBL gene and RUNX1 gene may cooperate in the pathogenesis of CMML in patients with FPD/AML. The presence of CBL mutations and 11q-aUPD was an important “second hit” that could be an indicator of leukemic transformation of MDS or AML in patients with FPD/AML.

Summary Mutations in the colony-stimulating factor 3 receptor (CSF3R) and calreticulin (CALR) genes have been reported in a proportion of adults with myeloproliferative disease. However, little is known about CSF3R or CALR mutations in paediatric myeloid disorders. We analysed CSF3R exons 14 and 17, and CALR exon 9, using direct sequencing in samples of paediatric acute myeloid leukaemia (AML; n = 521), juvenile myelomonocytic leukaemia (JMML; n = 40), myelodysplastic syndrome (MDS; n = 20) and essential thrombocythaemia (ET; n = 21). CSF3R mutations were found in 10 (1·92%) of 521 patients with AML; two in exon 14 (both missense mutations resulting in p.T618I) and eight in exon 17 (three frameshift mutations: p.S715X, p.Q774R, and p.S783Q; and five novel missense mutations: p.Q754K, p.R769H, p.L777F, p.T781I, and S795R). All of the patients with mutations in CSF3R exon 17 had chromosomal translocations, including four with t(8;21). At the time of reporting, seven of these ten patients are alive; three have died, due to side effects of chemotherapy. No CSF3R mutations were found in cases of MDS, JMML or ET. The only mutation found in the CALR gene was a frameshift (p.L367 fs) in one ET patient. We discuss the potential impact of these findings for the leukaemogenesis and clinical features of paediatric myeloid disorders.

Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domain was invariably affected. Our results highlight the role of RNase IIIb domain mutations in DICER1 along with TP53 inactivation in PPB pathogenesis. Cancer Res; 74(10); 2742–9. ©2014 AACR.

Introduction Pediatric acute myeloid leukemia (AML) comprises less than 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Approximately 40% of patients still have a relapse after first-line therapies, and the expected long-term survival rate decreases following relapse. Stem cell transplantation (SCT) was a conclusive strategy for de novo AML patients with a high risk and relapsed or refractory patients with standard and intermediate risk in a recent clinical trial. AML is a molecularly and clinically heterogeneous disease caused by various genetic alterations. Thus, it is difficult to accurately evaluate risk stratification even if known representative molecular markers, including KIT, FLT3-ITD, t(8;21)/RUNX1-RUNX1T1, and KMT2A (also known as MLL)-rearrangements, are used. Methods We investigated differences in the genetic background between SCT and non-SCT groups in participants of the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Among 369 patients with de novo AML, 175 patients received SCT. A standardized form was used to record clinical variables, including patient demographic information. The clinical data of patients in each risk group were followed for 3 years after the date of final registration. JPLSG performed a central review of morphologic classification and karyotyping based on the World Health Organization Classification, French-American-British classification, and cytogenetic analysis using conventional G-banding. Molecular characterization included mutational analyses of KIT (exons 8 and 17), N- and K-RAS (exons 1 and 2), NPM1 (exon 12), CEBPA (exon 12), FLT3-ITD, NUP98-NSD1,and CBFA2T3-GLIS2 gene rearrangement, as well as KMT2A- partial tandem duplication (MLPA methods). We also evaluated the gene expression of MECOM (also known as EVI1) and PRDM16 (also known as MEL1) because their high expressions are known poor prognostic markers. Overall survival (OS) was defined as the time from AML diagnosis to death or censorship at the last follow-up. Event-free survival (EFS) was defined as the time from AML diagnosis to treatment failure, relapse, death, or last follow-up. Results The 3-year OS among SCT patients (n = 175) was approximately 50%. It was significantly worse than that of non-SCT patients (n = 194, 90%; P < 0.001). Among 137 CBF-AML patients, 44 patients (32%) received SCT and their 3-year OS was 80%. On the other hand, among 232 non-CBF-AML patients, 131 patients (57%) received SCT, and their 3-year OS was only 35%. This result indicated that SCT is beneficial for relapsed CBF-AML, whereas most non-CBF patients who received SCT did not obtain the clinical benefit. In terms of the molecular characteristics, among 58 patients with high EVI1 expression, 39 patients (67%) received SCT, and their 3-year OS was approximately 35%. EVI1 expression was especially useful when using with MLL rearrangement because prognosis of patients with both MLL rearrangement andhigh EVI1 expression were extremely poor. On the other hand, among 84 patients with high PRDM16 expression, 63 patients (75%) received SCT, and their 3-year OS was approximately 20%. Although all AML patients with FLT3-ITD were assigned to receive SCT in the AML-05 trial, FLT3-ITD(+) patients withlow PRDM16 expression had a better outcome than FLT3-ITD(+) patients with high PRDM16 expression (3-year OS: 70% vs. 21%; P < 0.001). This result indicated that FLT3-ITD itself might not necessarily be associated with poor prognosis. PRDM16 gene expression is a useful marker to select patients needing SCT, particularly for patients with FLT3-ITD. Conclusion SCT was beneficial for patients with CBF-AML, whereas SCT was insufficient to rescue patients with non-CBF AML who relapsed, particularly patients with both FLT3-ITD(+) and high PRDM16 expression. New strategies, such as gemtuzumab ozogamicin or haploidentical SCT, are urged to rescue high risk/refractory patients. Disclosures No relevant conflicts of interest to declare.

Background: Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with a current overall survival rate of less than 70%. The pathogenesis of AML is heterogeneous and can be caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations [e.g., t(8;21), inv(16), and MLL-rearrangements] have been well established as diagnostic and prognostic markers of AML. Furthermore, recurrent mutations in FLT3, KIT, NPM1, and CEBPA have been reported in both adult and pediatric AML. Recently, massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for the prediction of the prognosis. However, these mutations are rare in pediatric AML, suggesting that other genetic alterations exist in pediatric AML. In contrast, recent reports have described NUP98-NSD1 fusion as an adverse AML prognostic marker and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harboring NUP98-NSD1 fusion. Intriguingly, PRDM16 overexpression occurs in nearly one-quarter of all children, with AML involving NUP98-NSD1-negative patients. Moreover, this overexpression is enriched in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1, and MLL-PTD). Patients and Methods: To reveal a complete registry of gene rearrangements and other genetic lesions in pediatric AML with a normal karyotype, we performed transcriptome analysis (RNA sequencing) of 61 of 70 de novo pediatric AML patients with a normal karyotype using Illumina HiSeq 2000. We could not perform RNA sequencing in nine patients because of a lack of RNA quantity or quality. Among the 70 AML patients with a normal karyotype, 33 patients overexpressed PRDM16, which was found to be strongly associated with a poor prognosis in our previous studies. All patients were enrolled and treated with AML-05 in the study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG). We also analyzed the known genetic mutations associated with these patients using the data derived from RNA sequencing. Results: A total of 144 candidate gene rearrangements, which were not observed in normal samples, were identified in 51 of 61 samples. Many of the recurrent gene rearrangements identified in this study involved previously reported targets in AML, including NUP98-NSD1, NUP98-JARID1A, CBFA2T3-GLIS2, MLL-MLLT10, and MLL-MLLT3. However, several gene rearrangements were newly identified in the current study, including MLL-SEPT6, HOXA10-HOXA-AS3, PRDM16-SKI, and CUL1-EZH2. We have also performed the validation of these novel gene rearrangements using Sanger sequencing. Most of these gene rearrangements were found in patients with a high expressionof PRDM16. In contrast, CEBPA mutations were frequently observed in patients with a low expression of PRDM16. Known gene alterations, such as FLT3-ITD and MLL-PTD, and mutations of the RAS, KIT, CEBPA, WT1, and NPM1genes were also detected using RNA sequencing. Conclusion: RNA sequencing unmasked a complexity of gene rearrangements and mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from adult counterparts, regarding the spectrum of gene rearrangements and mutations. In the present study, we identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including some novel fusion genes. These findings suggest that gene rearrangements in conjunction with mutations also play essential roles in pediatric AML. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

[Background] Pediatric acute myeloid leukemia (AML) remains to be a disease with a poor prognosis, with a long-term survival rate of approximately 60%. We have recently reported that high PRDM16 expression (high-PRDM16) was common in t(5;11)(q35;p15.5)/NUP98-NSD1-positive patients, and that high-PRDM16 was found to be a novel poor prognostic factor in this disease. However, this finding was not observed in FAB-M7. The presence of inv(16)(p13.3q24.3)/CBFA2T3-GLIS2 was postulated to be a reason. This fusion was reported as the most frequent fusion gene associated with a poor prognosis in FAB-M7, and we found that all CBFA2T3-GLIS2-positive patients had a low PRDM16 expression (low-PRDM16). Furthermore, some studies reported that bone morphogenetic protein 2 (BMP2) was frequently and highly expressed in CBFA2T3-GLIS2-positive patients, indicating the importance of BMP2 in pathogenesis in a subset of AML. In this study, we analyzed the expression status of BMP2 and PRDM16 and their relationship to prognosis in pediatric AML. [Patients and Methods] A total of 369 AML patient samples, who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial, were analyzed. Expression level of BMP2 and PRDM16 is analyzed by real-time quantitative PCR (Taqman Gene Expression Assay), using ABL1 as a control gene. The cutoff value for high-BMP2 was defined as the lowest expression level among 11 CBFA2T3-GLIS2-positive patients, and that for high-PRDM16 was defined as the lowest expression level among 10 NUP98-NSD1-positive patients. We also screened fusion genes and gene mutations of CBFA2T3-GLIS2, NUP98-JARID1A, RBM15-MKL1, MLL rearrangements, NUP98-NSD1, FUS-ERG, DEK-NUP214, RUNX1-RUNX1T1, CBFB-MYH11, and gene mutations of KIT, NRAS, KRAS, WT1, NPM1, MLL-PTD, and FLT3-ITD. [Results] Out of 369 patients, high-BMP2 and high-PRDM16 were identified in 47 (12.7%) and 44 (11.9%) patients, respectively. Only three (0.8%) of 369 patients had both high-BMP2 and high-PRDM16 (high-BMP2/PRDM16). In 44 high-BMP2 patients and 41 high-PRDM16 patients (3 high-BMP2/PRDM16 patients were excluded), MLL rearrangements were only found in high-BMP2 patients (25.5% vs 0%, p < 0.001), whereas FLT3-ITD was significantly much more frequent in high-PRDM16 patients (4.5% vs 56.1%, p < 0.001). Core-Binding-Factor (CBF)-AML was rare in both groups (4.5% and 0%, p = 0.495). Kaplan|Meier analyses revealed that high-BMP2 patients and high-PRDM16 patients had a low 5-year overall survival (OS) (46.3% and 29.3%, respectively). In molecular subgroup analyses, the prognosis of MLL-rearranged patients, including 12 high-BMP2 patients (21.8%), was not influenced by BMP2 expression (5-year OS: high-BMP2 66.7% vs low-BMP2 64.9%, p = 0.488), suggesting that the prognosis of MLL-rearranged patients might primarily be defined by their fusion partner genes and other gene aberrations as previously reported (e.g., EVI1 expression). Considering this result, further survival analyses of the patients lacking CBF-AML or MLL-rearrangements were performed (n = 174, 5-year OS: 50.5%). In this subgroup, 5-year OS of high-BMP2 patients (n = 30) and high-PRDM16 patients (n = 41) were significantly lower than that of low-BMP2/low-PRDM16 patients (36.7% vs 67.2%, p < 0.001, and 29.3% vs 67.2%, p < 0.001, respectively). [Conclusions] High-BMP2 and high-PRDM16 could identify distinct subgroups with poor prognosis in pediatric AML. Further investigation of molecular-targeted therapy against these genes may contribute to the improvement of clinical outcome in these subgroups. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome (DS) is characterized by the transient appearance of blast cells, which resolves spontaneously. Approximately 20 % of patients with TAM die at an early age due to organ failure, including liver disease. We studied 25 DS-TAM patients retrospectively to clarify the correlation between clinical and laboratory characteristics and liver diseases. Early death (

Background. Mutations in the ATP6V1B1 and the ATP6V0A4 genes cause primary autosomal-recessive distal renal tubular acidosis (dRTA). Large deletions of either gene in patients with dRTA have not been described. Methods. The ATP6V1B1 and ATP6V0A4 genes were directly sequenced in 11 Japanese patients with primary dRTA from nine unrelated kindreds. Large heterozygous deletions were analyzed by quantitative real-time polymerase chain reaction (PCR). The clinical features of the 11 patients were also investigated. Results. Novel mutations in the ATP6V1B1 gene were identified in two kindreds, including frameshift, in-frame insertion and nonsense mutations. Large deletions in the ATP6V0A4 gene were identified in two kindreds. Exon 15 of ATP6V0A4 was not amplified in one patient, with a long PCR confirming compound heterozygous deletions of 3.7and 6.9-kb nucleotides, including all of exon 15. Direct DNA sequencing revealed a heterozygous frameshift mutation in ATP6V0A4 in another patient, with quantitative real-time PCR indicating that all exons up to exon 8 were deleted in one allele. Clinical investigation showed that four of the six patients with available clinical data presented with hyperammonemia at onset. Conclusions. To our knowledge, these dRTA patients are the first to show large deletions involving one or more entire exons of the ATP6V0A4 gene. Quantitative PCR amplification may be useful in detecting heterozygous large deletions. These results expand the spectrum of mutations in the ATP6V0A4 and ATP6V1B1 genes associated with primary dRTA and provide insight into possible structure–function relationships.

The cryptic t(5;11)(q35;p15.5) creates a fusion gene between the NUP98 and NSD1 genes. To ascertain the significance of this gene fusion, we explored its frequency, clinical impact, and gene expression pattern using DNA microarray in pediatric acute myeloid leukemia (AML) patients. NUP98-NSD1 fusion transcripts were detected in 6 (4.8%) of 124 pediatric AML patients. Supervised hierarchical clustering analyses using probe sets that were differentially expressed in these patients detected a characteristic gene expression pattern, including 18 NUP98-NSD1-negative patients (NUP98-NSD1-like patients). In total, a NUP98-NSD1-related gene expression signature (NUP98-NSD1 signature) was found in 19% (24/124) and in 58% (15/26) of cytogenetically normal cases. Their 4-year overall survival (OS) and event-free survival (EFS) were poor (33.3% in NUP98-NSD1-positive and 38.9% in NUP98-NSD1-like patients) compared with 100 NUP98-NSD1 signature-negative patients (4-year OS: 86.0%, 4-year EFS: 72.0%). Interestingly, t(7;11)(p15;p15)/NUP98-HOXA13, t(6;11)(q27;q23)/MLL-MLLT4 and t(6;9)(p22;q34)/DEK-NUP214, which are known as poor prognostic markers, were found in NUP98-NSD1-like patients. Furthermore, another type of NUP98-NSD1 fusion transcript was identified by additional RT-PCR analyses using other primers in a NUP98-NSD1-like patient, revealing the significance of this signature to detect NUP98-NSD1 gene fusions and to identify a new poor prognostic subgroup in AML.

Mutations in Wilms tumor 1 (WT1) have been reported in 10–22 % of patients with cytogenetically normal acute myeloid leukemia (CN-AML), but the prognostic implications of these abnormalities have not been clarified in either adults or children. One hundred and fifty-seven pediatric AML patients were analyzed for WT1 mutations around hotspots at exons 7 and 9; however, amplification of the WT1 gene by the reverse transcriptase-polymerase chain reaction was not completed in four cases (2.5 %). Of the 153 evaluable patients, 10 patients (6.5 %) had a mutation in WT1. The incidence of WT1 mutations was significantly higher in CN-AML than in others (15.2 vs. 4.5 %, respectively, P = 0.03). Of the 10 WT1-mutated cases, eight (80 %) had mutations in other genes, including FLT3-ITD in two cases, FLT3-D835 mutation in two, KIT mutation in three, MLL-PTD in three, NRAS mutation in one, and KRAS mutation in two (in some cases, more than one additional gene was mutated). The incidences of KIT and FLT3-D835 mutations were significantly higher in patients with than in those without WT1 mutation. No significant differences were observed in the 3-year overall survival and disease-free survival; however, the presence of WT1 mutation was related to a poor prognosis in patients with CN-AML, excluding those with FLT3-ITD and those younger than 3 years.

Recently, bone marrow transplantation from an unrelated donor has been recommended as an option for the treatment of very severe aplastic anemia (vSAA) refractory to immunosuppressive therapy (IST) in the absence of a human leukocyte antigen-matched related donor (MRD). For SAA patients with complications such as bacterial infections, prompt transplantation using either a mismatched related donor or cord blood (CB) becomes necessary. However, the former option is associated with graft-versus-host disease, whereas the latter option is associated with a more significant risk of graft failure. We report a patient with vSAA refractory to IST that was repeatedly complicated by bacterial infection. An MRD was unavailable for this patient, so we decided on emergent transplantation and successfully performed CB transplantation (CBT) using a low-dose TBI conditioning regimen. Although it may be necessary to examine more clinical cases in the future, a conditioning regimen such as that involving low-dose TBI may decrease the risk of graft failure, and CBT may become a treatment option for vSAA in the absence of a suitable donor.

The cryptic t(5;11)(q35;p15.5) creates a fusion gene between the NUP98 and NSD1 genes. To ascertain the significance of this gene fusion, we explored its frequency, clinical impact, and gene expression pattern using DNA microarray in pediatric acute myeloid leukemia (AML) patients. NUP98-NSD1 fusion transcripts were detected in 6 (4.8%) of 124 pediatric AML patients. Supervised hierarchical clustering analyses using probe sets that were differentially expressed in these patients detected a characteristic gene expression pattern, including 18 NUP98-NSD1-negative patients (NUP98-NSD1-like patients). In total, a NUP98-NSD1-related gene expression signature (NUP98-NSD1 signature) was found in 19% (24/124) and in 58% (15/26) of cytogenetically normal cases. Their 4-year overall survival (OS) and event-free survival (EFS) were poor (33.3% in NUP98-NSD1-positive and 38.9% in NUP98-NSD1-like patients) compared with 100 NUP98-NSD1 signature-negative patients (4-year OS: 86.0%, 4-year EFS: 72.0%). Interestingly, t(7;11)(p15;p15)/NUP98-HOXA13, t(6;11)(q27;q23)/MLL-MLLT4 and t(6;9)(p22;q34)/DEK-NUP214, which are known as poor prognostic markers, were found in NUP98-NSD1-like patients. Furthermore, another type of NUP98-NSD1 fusion transcript was identified by additional RT-PCR analyses using other primers in a NUP98-NSD1-like patient, revealing the significance of this signature to detect NUP98-NSD1 gene fusions and to identify a new poor prognostic subgroup in AML.

Trib1 has been identified as a myeloid oncogene in a murine leukemia model. Here we identified a TRIB1 somatic mutation in a human case of Down syndrome–related acute megakaryocytic leukemia. The mutation was observed at well-conserved arginine 107 residue in the pseudokinase domain. This R107L mutation remained in leukocytes of the remission stage in which GATA1 mutation disappeared, suggesting the TRIB1 mutation is an earlier genetic event in leukemogenesis. The bone marrow transfer experiment showed that acute myeloid leukemia development was accelerated by transducing murine bone marrow cells with the R107L mutant in which enhancement of ERK phosphorylation and C/EBPα degradation by Trib1 expression was even greater than in those expressing wild-type. These results suggest that TRIB1 may be a novel important oncogene for Down syndrome–related acute megakaryocytic leukemia.

Background Several molecular markers, such as FLT3-internal tandem duplication (ITD), NPM1, CEBPA are well known to correlate with mortality in patients with acute myeloid leukemia (AML). Recently, a number of gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3. However, DNMT3A, IDH1/2, and TET2 are rare in pediatric patients with AML, thus accurate risk evaluation remained challenging even after incorporating these molecular markers. On the other hand, overexpression of the EVI1 gene is reported to be associated with adverse outcome in pediatric AML. Moreover, we have previously reported that measuring of PRDM16 gene expression was a powerful tool to predict the prognosis of pediatric AML. PRDM16 gene is highly homologous to the MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 gene. In this study, we investigated EVI1 gene expression to verify the prognosis of EVI1 gene expression and the relationship between EVI1 and PRDM16 gene expression. Methods Between 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 116 patients were excluded from the study because of misdiagnosis and unavailability of their RNA samples. Therefore, 369 patients were analyzed. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of EVI1 and association between EVI1 and PRDM16 genes. Results A total of 58 of 369 patients (15.7%) showed high expression of EVI1 gene. Overexpression of EVI1 gene was strongly associated with dismal prognosis; low risk (LR; 1 of 123 patients, or 0.8%); intermediate risk (IR; 38 of 147 patients, or 25.9%); high risk (HR; 6 of 50 patients, or 12%); and non-complete remission (Non-CR; 13 of 49 patients, or 26.5%), (P < 0.001). Overexpression of EVI1 correlated with the following characteristics: younger age at diagnosis; M4, M5, and M7 subtype; higher coincidence of MLL-rearrangement; and lower coincidence of t(8;21), and inv(16). EVI1 overexpression was very frequent among patients with de novo pediatric AML and IR/non-CR groups. Furthermore, more than half of patients in M6 (5 of 8 patients, or 62.5%) were EVI1 high expression. Interestingly, no patients with EVI1 high expression in M7 had a fusion of CBFA2T3-GLIS2. Patients with EVI1 overexpression also more frequently harbored a complex karyotype and monosomy 7. The frequencies of patients with high or low EVI1 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 1% vs 99%, P < 0.001; inv(16), 0% vs 100%, P < 0.001; NUP98-JARID1A, 83% vs 17%, P < 0.001; OTT-MAL, 100% vs 0%, P = 0.02; and KIT, 5% vs 95%, P = 0.003. The overall survival (OS) and event-free survival (EFS) among patients with EVI1 high expressions were significantly lower than that among patients without such gene aberrant expression (3-year OS 54% vs. 77%, P=0.008G3-year EFS: 34% vs 58%, P On the other hand, a total of 84 of 369 patients (22.8%) showed high expression of PRDM16 gene. The OS and EFS among PRDM16 overexpressing patients were significantly worse than those among low expression group (3-year OS: 51% vs 81%, P Conclusions We investigated EVI1 and PRDM16 gene expression in de novo pediatric AML patients, and their high expression was associated with inferior survival, respectively. We suggest that high EVI1 and/or PRDM16 expression is useful marker for adverse outcome. On the other hand, low EVI1 and PRDM16 expressions are useful to elucidate low risk patients. Disclosures No relevant conflicts of interest to declare.

Background Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia cases. AML is a major therapeutic challenge in pediatric oncology, and the current overall survival rate is In addition, recent studies have reported that the NUP98-NSD1 fusion is an adverse AML prognostic marker and that PRDM16 (also termed MEL1) is a representative gene that is overexpressed in patients who have the NUP98-NSD1 fusion. PRDM16 overexpression occurs in nearly a quarter of pediatric AML patients who are NUP98-NSD1 negative, and this overexpression is increased in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1,and MLL-PTD). Patients and Methods To obtain a complete overview of gene rearrangements and other genetic lesions, we performed RNA sequencing of samples from 47 de novo pediatric AML patients using Illumina HiSeq 2000, including 39 patients with normal karyotypes and 6 patients with Trisomy 8. Among these 47 patients, 35 patients overexpressed PRDM16, which was strongly associated with a poor prognosis in our previous studies. As a control, we selected 12 patients with low PRDM16 expression levels. All patients were enrolled and treated as part of the AML-05 study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group. We determined the known gene mutations present in these patients using the RNA sequencing data. Results Approximately 300 candidate gene rearrangements were identified in 46/47 samples, including 26 in-frame and 78 out-of-frame gene rearrangements. Several recurrent gene rearrangements identified in this study involved previously reported targets in AML, such as FUS-ERG, NUP98-NSD1,and MLL-MLLT3. However, several novel gene rearrangements were identified in the current study, including HOXA10-HOXA-AS3, PRDM16-XXX, CUL1-YYY, and DAZAP1-ZZZ. At present, we are validating these novel gene rearrangements using Sanger sequencing. Known gene alterations, such as FLT3-ITD, MLL-PTD, and mutations of RAS, KIT, CEBPA, WT1, and NPM1 genes, were detected by RNA sequencing. Conclusion In the present study, RNA sequencing was employed to elucidate the complexity of gene rearrangements/mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that can be discriminated from adult AML in terms of the spectrum of gene rearrangements/mutations. We identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including various novel fusion genes. Thus, our results suggest that gene rearrangements and mutations play essential roles in pediatric AML. Disclosures No relevant conflicts of interest to declare.

Myeloproliferative diseases (MPDs) in childhood are quite rare. Although pediatric and adult MPDs exhibit similar hematologic findings, JAK2V617F mutations and clonality status of MPDs in the DNA of neutrophils are evaluated less frequently in children than in adults. Increased incidence of venous thrombosis at uncommon sites is associated with JAK2V617F mutation in MPDs and thrombotic complications are more common in essential thrombocythemia (ET). Here, we describe 6-year-old girl with clonal myelopoiesis and JAK2V617F-positive ET associated with cerebral venous sinus thrombosis. To our knowledge, this is the first report of pediatric monoclonal and JAK2V617F-positive ET with cerebral venous sinus thrombosis.

Down syndrome (DS) patients are frequently complicated with infections, autoimmune phenomena and hematological disorders, including transient abnormal myelopoiesis (TAM) in infancy and acute megakaryoblastic leukaemia (AMKL) in later life. In this study, serum levels of cytokines from 23 TAM and 15 AMKL patients were examined using the highly sensitive microsphere fluorescence system. Statistical differences between DS neonates with or without TAM were found in IL-1beta [median 7.0 pg/ml (0.34-271.6) verses 0.05 pg/ml (0.0-2.4), p=0.034], TNF-alpha [8.11 pg/ml (0.1-253.0) verses 0.41 pg/ml (0.1-1.5), p=0.041], and IFN-gamma [20.0 pg/ml (0.14-406.3) verses 1.5 pg/ml (0.14-5.79), p=0.036]. Moreover, abnormal inflammatory cytokinemia was also found in myelodysplastic syndrome (MDS) and AMKL with DS. These abnormal cytokinemia may have a role in the pathophysiology of TAM, MDS and AMKL in DS, especially in liver fibrosis or myelofibrosis.

Background; Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of CEBPA,NPM1, DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3, because the recent development of massively parallel sequencing technologies. We have performed whole-exome sequencing of paired tumor-normal DNA from 19 patients, and identified 80 somatic mutations or 4.2 mutations per sample. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, BCORL1, EZH2, and major cohesin components including SMC3 and RAD21. In addition to these mutations, we also identified disease-associated candidate genes of ASXL2, PAX5 and others. Recently, recurrent somatic mutations in ASXL1 have been reported to occur in patients with adult AML, and to be associated with adverse outcome. Another study suggested that ASXL1 or ASXL2 mutations were associated with a high incidence of relapse. To reveal the significance of these mutations, we performed mutational analysis of ASXL1 and ASXL2 in 184 pediatric AML patients. Methods; Between 2006 and 2010, 485 de novo pediatric AML patients aged To estimate the frequency and prognostic impact of the ASXL1 and ASXL2 mutations in pediatric AML, we performed targeted sequencing of ASXL1 (exon 12) and ASXL2 (exon 12) genes using next-generation sequencer in 184 de novo AML patients including 51 patients with t(8;21). We validated the mutations in ASXL1 and ASXL2 by Sanger sequencing. Furthermore, we investigated the correlation among these mutations, other cytogenetic alterations and clinical characteristics. Results and Discussion; ASXL1 mutations were identified in 4 of 184 de novo pediatric AML patients (2.2 %) and all 4 ASXL1 mutation positive patients harbored t(8;21). Two of them relapsed, and one died after relapsed. On the other hand, ASXL2 mutations were identified in 10 of 184 de novo pediatric AML patients (5.4%) and 6 of them harbored t(8;21). Five of these 10 patients relapsed, and 2 of them died after relapsed. Especially, all of 4 patients without t(8;21) relapsed (2 in M5a and 2 with CBFA2T3-GLIS2 in M7), and 2 died after relapsed. Although only one ASXL2 patients with t(8;21) relapsed (1/6 or 17%), no ASXL2 positive patients with t(8;21) died. ASXL2 mutations were more observed in AML patients with t(8;21) (11.8%, 6/51), but not in 13 patients with inv(16). Overall survival of the patients with or without ASXL2 mutations were 80% and 66.7% (p=0.54), respectively. ASXL1 and ASXL2 mutations were mutually exclusive in this study. Conclusion; Ten of 184 patients (5.4%) had mutations of ASXL2 in pediatric AML, and the outcome of ASXL2 mutant patients with t(8;21) was favorable. Among the 51 pediatric AML patients with t(8;21), ASXL2 mutations were detected in 6 (11.8%) patients. All of them have been survived, suggesting that ASXL2 mutations in patients with t(8;21) may be associated with favorable prognosis in pediatric AML in contrast to adult AML. On the other hand, 4 (2.2%) of 184 patients had ASXL1 mutations, and all of them were t(8;21). In these 4 patients, 2 of them relapsed, and one died after relapsed. Although the number of patients is too small, ASXL1 mutations were not considered to be associated with favorable outcome. Both ASXL1 and ASXL2 mutations were detected at high frequency among pediatric AML patients with t(8;21) and mutual exclusive. As we consider that further study will be needed to clarify the significance of these mutations, we are now analyzing mutations in other exons of ASXL2, and would like to report these data in the annual meeting. Disclosures No relevant conflicts of interest to declare.

Background Acute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes, leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Although, many prognosis-associated gene alterations have been identified in adult AML, genetic alterations responsible for an adverse outcome in most patients with pediatric AML have remained obscure.In our previous study, we have identified NUP98-NSD1 fusion gene in 6 of 124 pediatric AML patients. As a result of gene expression profile using microarray, all these 6 patients have showed high expression of HOXA9, HOXA10, HOXB3, HOXB5, HOXB6, and a zinc finger transcription factor gene MDS1-EVI1-like-1 (MEL1), and clustered a distinct poor prognostic subgroup [4-year-overall survival (OS); 33%]. Furthermore, this subgroup has also formed the bigger poor prognostic subgroup involving the 18 patients without NUP98-NSD1 fusion gene. We defined this subgroup as NUP98-NSD1 signature consisting of 24 of 124 patients (19%), 4-year-OS; 37.5%. MEL1 gene expression represented the specific characteristics of this subgroup.To verify the significance of MEL1 gene expression and the relationship with other poor prognostic markers such as FLT3-ITD, MLL-PTD, and NPM1, we investigated MEL1 expression by real-time PCR in 369 pediatric AML cases other than acute promyelocytic leukemia (APL) and Down syndrome-associated AML, because these subtypes formed genetically different unique entities and treated by other protocols. Interestingly, MEL1 is highly homologous to MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 genes. As EVI1 high expression has been reported as a poor prognostic marker in pediatric AML patients, especially in patients with MLL rearrangement, we also measured and examined the EVI1 gene expression. Methods Between 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 369 samples were available in this study. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and MEL1, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of MEL1 gene. Results A total of 84 of 369 patients (22.8%) showed high expression of MEL1 gene. The number of patients with MEL1 high expressions were gradually enriched in order of the intermediate risk (IR; 34 of 142 patients, or 23.9%), high risk (HR; 21 of 48 patients, or 43.8%), and non-complete remission (non-CR; 20 of 39 patients, or 51.3%), but were almost all absent in patients with a low risk (LR) cytogenetic profile (4 of 120 patients, or 3.3%) consisting of t(8;21) and inv(16). The absolute values of MEL1 gene expression have also gradually increased in order of LR < IR < HR < non-CR. The overall survival among patients with MEL1 high expressions was significantly lower than that among patients without such gene aberrant expression (50% vs. 82%, P Conclusions MEL1 high expression was highly recurrent in de novo pediatric AML patients with high/intermediate-risk cytogenetic profiles and was independently associated with a poor outcome. Combined with MEL1expression and genetic alterations enable to clarify the genetic background of pediatric AML profiles. The data in this study showed a way in which integrated mutational profiling of a clinical trial cohort can advance our understanding of the biologic characteristics of AML, improve current prognostic models, and inform prospective therapeutic decisions. Disclosures No relevant conflicts of interest to declare.

Background: Although pediatric acute myeloid leukemia (AML) still has a poor prognosis, advanced risk-stratified chemotherapy and effective supportive care have improved the outcome, with the long-term survival rate of 60–70%. Comprehensive molecular analysis using next-generation sequencing identified the heterogeneity of this disease, suggesting that it would be difficult to increase the effectiveness of risk stratification. FLT3-ITD, accounting for 12–15% of pediatric AML, is a well-known predictor of poor prognosis and is used for risk stratification. However, recent studies have reported frequent co-occurrence of FLT3-ITD with other prognostic factors such as NUP98-NSD1, MLL-PTD, NPM1, and DEK-NUP214. These findings prompted us to reconsider the significance of FLT3-ITD and search for other factors that can identify a poor-prognosis subgroup in pediatric AML patients with FLT3-ITD. Patients and Methods: From 2006 to 2010, 485 de novo pediatric AML patients aged Results and Discussion: Survival analysis in FLT3-ITD-positive patients (n = 47) revealed that patients with FAB subtypes M4 and M5 (M4/5 patients; n = 9 and 11, respectively) had a much poorer prognosis than those with other FAB subtypes (non-M4/5 patients; n = 27; 4-year pOS: 7.5% vs 70.3%, p < 0.0001). Six of 9 FAB-M4 patients and all FAB-M5 patients died, indicating that the M4/5 patients are strongly associated with a poor survival in FLT3-ITD-positive patients. In M4/5 patients (n = 20), age was not statistically different (11.3 years vs 10.9 years, p = 0.5902), but the WBC count was significantly higher than non-M4/5 patients (110,200/ µL vs 26,200/ µL, p = 0.0176). In M4/5 patients, NUP98-NSD1 (n = 6), MLL-PTD (n = 3), DEK-NUP214 (n = 2), KIT mutation (n = 2), and WT1 mutation (n = 3) were repeatedly detected, and all patients with any of these aberrations died. Prognostic independence of NUP98-NSD1 and MLL-PTD remains to be determined because they are associated with a high incidence of FLT3-ITD. Meanwhile, 6 patients did not have any molecular aberrations, and 4 patients had a normal karyotype, suggesting the presence of cryptic fusion genes or unknown gene mutations. On the other hand, favorable genetic aberrations were frequent in non-M4/5 patients (n = 27). RUNX1-RUNX1T1 and NPM1 mutations were found in 3 and 5 non-M4/5 patients, respectively. All of these patients are still alive without a relapse, suggesting that the effects of FLT3-ITD are reversed by some favorable genetic aberrations, although the underlying mechanism remains unknown. Non-M4/5 patients who died (n = 7) included 1 patient each with NUP98-NSD1 and CBFA2T3-GLIS2; thus, further criteria are necessary to not misclassify these patients. Although it is unclear why the poor prognosis associated with M4/5 was not noticed to date, the possibilities are racial difference and the small sample size of Japanese cohort studies. In addition, the problem is that there are no promising treatments at present for patients with a very poor outcome, even if such a distinct subgroup is identified. Conclusions: We identified the prognostic significance of the FAB classification which stratified Japanese pediatric AML patients with FLT3-ITD into a very high-risk group and intermediate-risk group. The poor prognosis associated with NUP98-NSD1, MLL-PTD, and DEK-NUP214 may be worsened with the co-occurrence of FLT3-ITD and FAB-M4/5. We believe that these findings will help to develop not only a risk-stratified therapy, but also an epoch-making treatment for this poor-prognosis subgroup of pediatric AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.