ALBERT

Description: Abstract 2536 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 important molecular markers have already been discovered in AML, no known prognosis-associated cytogenetic aberrations or mutations were detectable in a subset of AML patients. In this regard, recent reports of somatic mutations affecting the C-terminal zinc finger (ZF) 2 of GATA2 are intriguing, because these GATA2 mutations were associated with the progression of chronic myeloid leukemia, whereas hereditary ZF2 of GATA2 mutations predispose to AML and myelodysplastic syndrome. GATA2 mutations were also reported as a predisposing gene of the monocytopenia, mycobacterial infection (MonoMAC) syndrome and the dendritic cell, monoctye, B and NK lymphoid deficiency (DCML) syndrome. GATA2 belongs to a family of zinc finger transcription factors, and is important for hematopoietic stem cell proliferation and normal megakaryocytic development. These findings prompted us to search for possible GATA2 mutations in pediatric AML. Methods To explore the frequency and clinical impact of GATA2 mutations, we examined 157 Japanese pediatric AML patients, including 13 with FAB-M3 and 10 with Down syndrome (DS) who were treated on different treatment protocol, by PCR following direct sequencing. As GATA2 mutations thus far reported almost exclusively involved exons 4–6 that encode zinc finger 1 and 2 domain, we confined our analysis to these exons. Results GATA2 missense mutations were found in 7 out of 157 patients (4.5%). Notably, All of GATA2 mutations were located in ZF2 in this study, although almost all of GATA2 mutations in adult AML were located in ZF1. Wild type GATA2 were found in 3 of 3 AML patients with GATA2 mutation. The other 4 patients had no history of Mono MAC syndrome, suggesting that these mutations were acquired. The zinc finger region of GATA2 is required for binding to promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA2 and can modify its transactivation capacity. Interestingly, 2 GATA2 mutations were found in FAB-M3 in this study, GATA2 mutations also may be associated with acute promyelocytic leukemia. Clinical and molecular features between patients with and without GATA2 mutations were not significantly different in the clinical parameters (WBC, age, sex, etc.), and the outcome of GATA2 mutation positive patients was not poor when compared to GATA2 mutation negative patients: Two of the 7 patients received allogeneic-stem cell transplantation (Allo-SCT) and one of them died of gastrointestinal hemorrhage after SCT. The other 5 patients who did not receive the SCT were still alive. Conclusion GATA2 is a new predisposition gene for pediatric AML and shows functional changes caused by mutations within a highly conserved threonine repeat located in ZF2. Interestingly, MonoMAC and DCML syndromes were not observed in de novo AML patients with GATA2 mutations. Although further investigation is needed, our results indicated that GATA2 mutations were associated with a favorable outcome in pediatric AML. Most of the patients with GATA2 mutations have been classified into an intermediate risk group in our study, however, their favorable outcome suggests that less aggressive treatment strategy without SCT might be appropriate for AML patients carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2149 Recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to results in aberrant tyrosine kinase signaling, which would lead also to activation of RAS signaling pathways. We and others reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and MDS/myeloproliferative neoplasm, especially in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The importance of CBL mutations concerning leukemogenesis is substantially increased. We investigated CBL mutations in 20 therapy-related leukemia/MDS (t-Leuk/MDS) cases and 26 infant leukemia cases. Homozygous mutation of theCBL gene (P417R), which was located in the RING finger domain, was identified in one out of 20 (5%) t-Leuk/MDS cases. This patient was a 5 year-old boy, whose biopsied specimen of the buccal lymph node showed malignant lymphoma (diffuse large T cell type, MT1(+), MB1(-), UCHL1(+)). No pathogenic nucleotide changes were identified in the CBL gene in the initial sample. Subsequently, the patient was treated with chemotherapy including VP-16 (200 mg/m2) given twice weekly. Nineteen months after initial diagnosis, he was diagnosed as having therapy-related leukemia with t(5;21) and MLL gene rearrangement due to VP-16. Furthermore, CBL gene mutations were found in 3 of 26 (12%) infant leukemia cases with 11q23 translocation/MLL gene rearrangement. CBL gene mutations were located in splice site in intron 8 and the RING finger domain (Y371H, in 2 cases), SNP array analysis (Affymetrix, GeneChip) of these cases with mutated CBL gene disclosed 11q-acquired uniparental disomy in all cases, but not in cases with wild-type CBL. CBL mutation has not been reported in acute leukemia with 11q23 translocation/MLL gene rearrangement. To our knowledge, these are the first t-Leuk/MDS case and infant cases with 11q23 translocation/MLL rearrangement, suggesting that CBL is mutated in a unique subset of t-Leuk/MDS and infant leukemia which is considered to play a pathogenic role in the development of t-Leuk/MDS and infant leukemia. Further accumulation of cases with t-Leuk/MDS and infant leukemia having the CBL mutation is needed. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: 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

Description: 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

Description: Abstract 1391 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. Many important molecular markers have already been discovered in AML that have not only helped to better characterize patients, but also helped to improve risk stratification. However, in a subset of AML patients, no known prognosis-associated cytogenetic aberrations or mutations were detectable. In hematological malignancies, 11p15 translocations involving the nucleoporin 98-kDa (NUP98) protein gene are relatively rare, but more than 20 different chromosomal rearrangements have been identified. The cryptic t(5;11)(q35;p15.5), which is frequently accompanied by deletion of the long arm of chromosome 5, del(5q), creates a fusion gene between NUP98 and the nuclear receptor-binding SET-domain protein 1 (NSD1) gene. Recently, Hollink et al identified NUP98-NSD1 gene fusion by high-resolution genome-wide copy number analysis and reverse transcription (RT)-PCR in pediatric and adult AML patients, and reported that NUP98-NSD1 gene fusion was a predictor of poor prognosis. Thus, we explored the frequency, gene expression pattern, and clinical impact of NUP98-NSD1 in a large cohort of Japanese pediatric AML patients. Methods From January 2000 to December 2002, 318 patients were newly diagnosed with de novo AML. Of these, samples from 124 patients whose gene expression profiling data were available were analyzed, including 10 patients with FAB-M3 and 6 patients with Down syndrome (DS) who were treated on different treatment protocols. For the detection of NUP98-NSD1 gene fusion, direct sequencing was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Gene expression profiling data, which were performed before, were available for these 124 patients. Hierarchical clustering analysis was performed using Cluster and Tree View software. Result NUP98-NSD1 fusion transcript was detected in 6 (4.8%) of 124 pediatric AML patients and a characteristic gene expression pattern related to these patients was also detected. Further hierarchical clustering analysis using the 87 probe sets which were differentially expressed in 6 NUP98-NSD1-positive patients did not detect a simple cluster of 6 NUP98-NSD1-positive patients but a relatively large cluster including 18 NUP98-NSD1-negative patients. When compared with 100 NUP98-NSD1 signature-negative patients, the 24 NUP98-NSD1 signature-positive patients frequently had normal karyotype (62.5%), and del(9q) (8.3%), but did not have any favorable chromosomal translocations such as t(8;21), inv(16) and t(15;17). The frequencies of M4 and M5 subtypes (14/24, P 〈 0.001) were also higher than those in NUP98-NSD1 signature-negative patients. In addition, they frequently had FLT3-ITD (12/24, P 〈 0.001), MLL-PTD (7/24, P = 0.124), NPM1 (3/24, P = 0.007) and WT1 (5/24, P = 0.037) mutations. In total, NUP98-NSD1 related gene expression signature (NUP98-NSD1 signature) represented 19% (24/124) of total patients and 58% (15/26) of cytogenetically normal patients in our cohort. Their 4-year overall survival (OS) and event-free survival (EFS) were poor when compared to 100 NUP98-NSD1 signature-negative patients (37.5% vs. 86.0% and 37.5% vs. 72.0%). Conclusion Our results indicated that Patients with NUP98-NSD1 related gene expression signature had extremely poor clinical outcome, irrespective of the presence of NUP98-NSD1 gene fusion. Most of the patients displaying the NUP98-NSD1 signature have been classified into an intermediate risk group, but their unfavorable outcome suggests that a high risk group is a more suitable stratification. These results suggest the presence of a new poor prognostic subgroup which revealed the value of the NUP98-NSD1 signature as prognostic markers in pediatric AML. Although further investigation is necessary, we believe that our work contributes to improve the risk stratification in pediatric AML. Disclosures: No relevant conflicts of interest to declare.