Abstract: Background Children with Down syndrome (DS), which caused by an extra copy of chromosome 21, are predisposed to develop acute lymphoblastic leukemia (ALL). On the other hand, in non-DS children, acquisition of chromosome 21 gain is observed in 95% of hyperdiploid (HD) ALL, which is the most common cytogenetic abnormality pattern in childhood ALL. These may suggest that gain of chromosome 21 relates molecular pathogenesis of ALL. Genetic aberrations of RUNX1 locus on chromosome 21 including iAMP21 or t(12; 21)(p13; q22)/ETV6-RUNX1 were often found in pediatric ALL. While recent studies implicated that HMGN1 or DYRK1A on chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Compared with ALL of non-DS children, DS-ALL have uncommon genetic alterations such as mutations in JAK2 and RAS, mutations or overexpression of CRLF2. These suggest that DS-ALL may have unique biological features compared with ALL of non-DS children. Difference of biological basis between them may correlate to worse prognosis of DS-ALL. Although microarray transcript profiling provided some characteristic gene expression in DS-ALL, no study showed comprehensive transcriptome analysis in DS-ALL. Purpose This study was conducted to elucidate comprehensive transcriptomic landscape in DS-ALL and to reveal biological features through clustering by gene expression profiling. Methods Our cohort includes 72 pediatric B-cell precursor ALL samples (48 DS-ALL, 13 HD-ALL, and 11 euploid (EP) ALL). All HD-ALL samples gained chromosome 21. Three cases of DS-ALL and one case of EP-ALL had ETV6-RUNX1 rearrangement. We applied genome-wide analysis using whole-transcriptome sequencing (WTS) to 55 samples, Illumina 450k methylation array to 12 samples, and Illumina EPIC methylation array to 26 samples. For gene expression profiling, we conducted consensus clustering algorithms. Data from methylation arrays were normalized by beta-mixture quantile normalization and merged by common probes. Results Our consensus clustering analysis of gene expression stratified samples into three groups such as DS-ALL (cluster 1), DS-ALL and HD-ALL (cluster 2), and EP-ALL (cluster 3). Eight cases with DS-ALL having RAS mutations were classified into cluster 1. DYRK1A, HMGN1, ETS2 on chromosome 21 were highly expressed in cluster 1 and cluster 2 compared with cluster 3. Expression of RUNX1 was lower in cluster 1 and cluster 2 although it was not significant. In addition, FLT3, ELK1, and ETV6 were more highly expressed in cluster 1 and cluster 2 compared with cluster 3. Comparing cluster 1 with cluster 2, NRAS, MAP2K1, JUN, FOS, WT1, EPOR, and PDGFRB were highly expressed in cluster 1. Methylation analysis indicated that methylation pattern of DS-ALL was distinct from that of HD-ALL. Using methylation analysis, we detected differentially methylated regions (DMRs) between DS-ALL and HD-ALL. In DMRs of promoter-associated regions, RUNX1, IGF2BP1, and ETV6 were included. These genes were significantly hypermethylated in DS-ALL. Next, we elucidated methylation profiling of DS-ALL compared with HD-ALL and found hypermethylated genes in DS-ALL. Although these genes may participate pathogenesis of DS-ALL, we could not find any association of gene expression pattern. Conclusion We identified specific gene expression profiling of DS-ALL and significantly upregulated genes in DS-ALL compared with HD-ALL and EP-ALL. Overexpression of these genes was characteristic gene profiling common to ALL with gaining extra chromosome 21. While ETS2, FLT3, ELK1, NRAS, MAP2K, JUN, FOS, and WT1 were highly expressed in AML, overexpression of these genes may also be related to pathogenesis of DS-ALL. In addition, DS-ALL highly expressed PDGFRB and EPOR, to which these genes are targetable by TKIs and may improve prognosis of DS-ALL. Intriguingly, RUNX1 was hypermethylated in DS-ALL compared with HD-ALL and EP-ALL, indicating that underexpression of RUNX1 was a unique gene profiling of DS-ALL and was due to hypermethylation of RUNX1. In DS patient, hyprmethylation of RUNX1 in their normal blood cells has been reported. This aberrant hypermethylation may be related to pathogenesis of DS-ALL. In this study, we unveiled gene expression profiling and methylation pattern of DS-ALL. However, to elucidate the whole picture of molecular pathogenesis of DS-ALL, we may need further analysis. Disclosures Kataoka: Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Abstract: Background Acute lymphoblastic leukemia (ALL) in Down syndrome (DS) have uncommon genetic alterations such as mutations of JAK2, RAS, and overexpressions of CRLF2. These findings suggest DS-ALL may have unique biological features compared with non-DS-ALL. While recent studies implicated HMGN1 or DYRK1A in chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Materials and Methods We performed whole transcriptome sequencing, targeted deep sequencing, and SNP array analysis in 25 DS-ALL samples, which included four ETV6-RUNX1 fusions and one high hyperdiploid. To compare with DS-ALL, we also performed whole transcriptome sequencing and whole exome sequencing to 118 non-DS-ALL samples, which included several subtypes such as ETV6-RUNX1 or BCR-ABL1. To cluster gene expression profiling, we applied the hierarchical clustering method. The detection of Ph-like signatures was performed by the hierarchical clustering by the gene set reported by Harvey. Results In expression analysis, we identified 19 fusions in 25 DS-ALL samples. These fusions included 15 recurrent fusions in pediatric BCP-ALL and 4 novel fusions, which including SSBP3-DHCR24, PDGFA-TTYH3, and NIN-NDUFA6. In novel fusions, PDGFA-TTYH3 fusions were detected in two DS-ALL samples. The hierarchical clustering analysis (Figure 1) combining 25 DS-ALL with 123 non-DS ALL samples. In our cohort, we defined samples with PAX5 alteration only such as a mutation or fusion as PAX5-altered. This clustering revealed ALL samples were divided into six clusters (cluster E1 to E6). Among six clusters, DS-ALL samples were divided into four clusters. In these four clusters, chi-square test revealed the significant enrichment of DS-ALL in E6 cluster. Importantly, our expression analysis revealed DS-ALL samples were highly heterogeneous and had the same expression pattern corresponding to each subtype same as non-DS-ALL. Cluster E3 included most samples with PAX5 fusions. All samples with ETV6-RUNX1 fusions were classified into cluster E4. Most samples of high hyperdiploid were classified into cluster E5. Cluster E6 was characterized by BCR-ABL1 fusions and Ph-like signatures. We detected 21 samples had Ph-like signatures, which included seven DS-ALL samples and 14 non-DS-ALL samples. Though we also analyzed differentially expressed genes between DS-ALL and non-DS-ALL, no genes on chromosome 21 such as HNGN1 or DYRK1A was significantly expressed. To investigate a relation between expression and genomic status, we further searched mutational analysis and copy number analysis (Figure 2). In 25 DS-ALL samples, six samples revealed JAK2 mutations and CRLF2 fusions. Interestingly, all of these six samples had Ph-like signatures. In cluster E5, one non-DS-ALL sample revealed JAK2 mutation and CRLF2 fusion and this particular sample was expected to have the Ph-like signature. To detect other Ph-like samples, we performed hierarchical clustering of 143 ALL samples based on the genes with a significantly (adjusted P value 〈 0.0001) high expression in already detected 21 Ph-like samples. This analysis revealed three additional samples (two DS-ALL and one non-DS-ALL) had Ph-like signatures. Intriguingly, Ph-like samples accounted for 36% in 25 DS-ALL samples. In contrast, because several subtypes in non-DS-ALL showed mutations of RAS pathway genes, mutations of RAS pathway genes are common drivers in pediatric BCP-ALL. Copy number analysis elucidated one DS-ALL sample in cluster E3 had a known focal amplification of chromosome 9 involving exon 2 to 5 of PAX5, which may result in dysfunction of PAX5. Though no report analyzed PAX5 status except for deletion in DS-ALL, DS-ALL had not only deletion of PAX5, but also miscellaneous aberrations such as amplification or fusion. One DS-ALL sample without ETV6-RUNX1 in cluster E4 had homozygous deletions of ETV6, implicating ETV6-RUNX1-like signature. Conclusion Our result suggested DS-ALL were highly heterogeneous. Though expression profiles of DS-ALL had similar to non-DS-ALL, frequencies of subtypes in DS-ALL were quite different from non-DS-ALL, that is, low incidence of ETV6-RUNX1 or HeH, and high incidence of Ph-like signatures. Because molecular targeting agents such as imatinib or ruxolitinib improve the prognosis of Ph-like ALL, these agents may be also promising for treatment of DS-ALL. Disclosures No relevant conflicts of interest to declare.

Abstract: Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML. Methods To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases. Results and Discussion A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%). Conclusion Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract: MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities that occur in acute myeloid leukemia (AML). Mutational landscapes in KMT2A-rearranged AML have been reported; however, most studies are missing data at relapse. Therefore, matched diagnostic and relapse samples were analyzed in this study, and the clonal evolution pattern in KMT2A-rearranged AML was examined. Further, the prognostic significance of the clonal architecture was investigated. Sixty-two diagnostic and 16 relapse samples obtained from pediatric patients with KMT2A-rearranged AML enrolled in the Japan Children's Cancer Group (JCCG) AML-05/AML-99 study were analyzed for 338 genes using targeted sequencing. The data were analyzed with the published data of 105 diagnostic and 9 relapse samples with KMT2A-rearranged AML. Additionally, as a control, the mutation data of matched diagnostic and relapse samples of 107 patients with non-KMT2A-rearranged AML were collected. Among 25 patients with KMT2A-rearranged AML with matched data at diagnosis and relapse, mutations of signaling pathway genes (FLT3, KRAS, NRAS, PTPN11, CBL, and BRAF) were frequently detected in diagnostic samples (25 mutations/25 patients). However, 21 of 25 (84.0%) mutations were lost at relapse. In contrast, 7 of 19 (36.8%) mutations of other pathway genes were lost at relapse, and the percentage was significantly lower than that of mutations in the signaling pathway genes (P = 0.002). Six mutations in the signaling pathway genes and 11 mutations in other pathway genes were acquired at relapse. Particularly, mutations of transcription factor genes (WT1, SPI1, GATA2, and RUNX1) were acquired at relapse: 7 of 8 (87.5%) mutations were detected only at relapse. These results suggest that mutations of signaling pathway genes are unstable in the clonal evolution of KMT2A-rearranged AML. Mutations of other pathway genes, especially those of transcription factor genes, may contribute to relapse in patients with KMT2A-rearranged AML. Next, attention was turned to the KRAS mutations (KRAS-MT) because we have previously shown that KRAS-MT are independent adverse prognostic factors in KMT2A-rearranged AML (Blood Adv. 2020). Among 25 patients with KMT2A-rearranged AML with matched data at diagnosis and relapse, 10 (40.0%) patients harbored KRAS-MT at diagnosis. Interestingly, KRAS-MT were lost at relapse in 9 of 10 (90.0%) patients. Among 107 patients with non-KMT2A-rearranged AML with matched data at diagnosis and relapse, 10 (9.3%) patients harbored KRAS-MT at diagnosis. The frequency of KRAS-MT was significantly higher in KMT2A-rearranged AML (40.0% vs. 9.3%, P = 0.0006). This may be explained on the basis of the fact that KRAS-MT is associated with a high relapse rate in KMT2A-rearranged AML, but not in non-KMT2A-rearranged AML. KRAS-MT was lost at relapse in 5 of 10 (50.0%) patients with non-KMT2A-rearranged AML. The percentage of KRAS-MT loss at relapse was higher in KMT2A-rearranged AML. However, it was not statistically significant (90.0% vs. 50.0%, P = 0.14). Therefore, KRAS-MT may be unstable in clonal evolution regardless of disease subtypes in AML. The underlying mechanisms of the paradox between the high relapse rate in patients with KRAS-MT and frequent loss of KRAS-MT at relapse in patients with KMT2A-rearranged AML should be examined in future studies. The loss of KRAS-MT at relapse suggests that the mutations were in subclones at diagnosis. Therefore, we finally examined the prognosis of 167 patients according to the clonality of KRAS-MT at diagnosis. In patients with KMT2A-MLLT3 (n = 67), those with subclonal KRAS-MT (n = 6) had adverse 5-y event-free survival compared with both patients with wild-type KRAS (KRAS-WT) (n = 56) (KRAS-WT vs. subclonal KRAS-MT: 58.7% vs. 16.7%, P = 0.04) and patients with clonal KRAS-MT (n = 5) (clonal KRAS-MT vs. subclonal KRAS-MT: 80.0% vs. 16.7%, P = 0.07). However, 5-y overall survival (OS) was similar among the three groups. In contrast, among patients with KMT2A-MLLT10 (n = 37), those with clonal KRAS-MT (n = 5) had adverse 5-y OS compared with both patients with KRAS-WT (n = 20) (KRAS-WT vs. clonal KRAS-MT: 59.7% vs. 0.0%, P = 0.006) and patients with subclonal KRAS-MT (n = 12) (subclonal KRAS-MT vs. clonal KRAS-MT: 58.3% vs. 0.0%, P = 0.04). According to these results, the effects of the clonality of KRAS-MT on prognosis may depend on which KMT2A fusion is present. Disclosures Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Saito: Toshiba corporation: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

Abstract: Introduction: In limited-stage diffuse large B-cell lymphoma (DLBCL), a continued risk of relapse has been reported (Stephens et al. J Clin Oncol 2016). Another report highlighted that initial limited-stage DLBCL, a favorable International Prognostic Index and extranodal involvement seemed to be risk factors for late relapse (LR; Larouche et al. J Clin Oncol 2010). However, even if a subgroup of patients (pts) with LR shows distinct clinical characteristics, the underlying biology is largely unknown. Methods: Nineteen consecutive pts who developed LR were identified among 337 pts with de novo DLBCL of Ann Arbor stage I/II who were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone), with/without rituximab, between 1997 and 2012. LR was defined as the first relapse of B-cell lymphoma (BCL; including indolent BCL) occurring more than 5 years after a primary diagnosis. The 19 pts underwent clinical, pathological (cell of origin according to the Hans algorithm) and genetic analyses. Genomic DNA (gDNA) was extracted from formalin-fixed, paraffin-embedded sections of tumor specimens at primary diagnosis and relapse, and from bone marrow (BM) specimens without tumor invasion as controls. Genomic DNA samples from paired primary and relapsed tumors were analyzed using BIOMED-2 multiplex PCR for immunoglobulin heavy chain (IGH) rearrangements (Invivoscribe Technologies, San Diego, CA, USA) to determine whether the paired tumors shared the same clonal IGH rearrangements. Target sequencing of 2709 regions across 69 lymphoma-related genes was performed on gDNA samples. Mutational calls were made in comparisons with individual control BM gDNA samples according to the criteria outlined in Table 1. All functional mutations detected in paired tumors per pt were compared, and shared and non-shared mutations were identified. Results: Baseline characteristics of the 19 pts were as follows: median age of 60 years (range, 32-77); 13 (68%) had stage I disease, and 11 (58%) had extranodal disease. At primary diagnosis, 14 of 19 pts (74%) had a non-germinal center B-cell-like (non-GCB) type DLBCL. As an initial treatment, CHOP therapy (median 4 cycles; range, 3-8), with (n=17) or without (n=2) radiation therapy, was performed in all 19 pts. Seven pts were treated with CHOP and rituximab. The median duration from initial diagnosis to LR was 8 years (range, 5-18). At LR, the median age was 71 years (range, 45-84). One pt relapsed as a composite of DLBCL and indolent BCL, and two pts relapsed as indolent BCL. Seven of the 19 pts were excluded from gene analysis because their paired gDNAs were of poor quality. The results of target sequencing combined with clinicopathological information and the results of IGH-PCR analysis are described in Table 2. Shared mutations between individual pairs of primary and relapsed tumors were detected in nine of the 12 pts analyzed. The most frequent shared mutation was a CD79B missense mutation of a single base substitution in positions 196 (n=5) or 199 (n=2). The second most frequent mutation was a MYD88 (L265P) missense mutation (n=5). Co-mutation of CD79B and MYD88 was found in four pts, who were considered to have a MCD (MyD88,CD79b) type DLBCL (Schmitz et al. N Engl J Med 2018). All eight pts with a CD79B and/or MYD88 mutation had a non-GCB type DLBCL. The same clonal IGH rearrangements between paired tumors were found in six of the eight pts. Seven of the eight pts presented with primarily extranodal disease originating from the testis (n=3), nasal/paranasal cavity (n=2) or gingiva (n=2). One pt (#9 in Table 2) with a GCB type DLBCL had shared mutations in MLL2, CREBBP, MEF2B and PIM1. However, different clonal IGH rearrangements were detected between paired tumors, implying these mutations may be the basis for lymphomagenesis; an on-going IGH rearrangement may occur. In the other three pts (#10-12), shared mutations were not detected. Mutations in TP53 were not detected in any pts. We did not identify any specific mutations considered to be associated with LR. Conclusions: Common characteristics shared in a subgroup of pts with limited-stage DLBCL who developed LR were as follows: non-GCB type, CD79B and/or MYD88 mutations, and extranodal disease at primary manifestation. The mechanisms of LR from the viewpoint of gene alterations were considered heterogeneous. Disclosures Maruyama: Asahi Kasei Pharma: Honoraria; Takeda: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Dai-ichi-Sankyo: Honoraria; Dai-Nippon-Sumitomo: Honoraria; MSD: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Fujifilm: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; AstraZeneca: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Amgen Astellas BioPharma: Research Funding; Otsuka: Research Funding; Novartis: Research Funding; Nippon Boehringer Ingelheim: Research Funding; Pfizer: Research Funding; Solasia Pharma: Research Funding; Celgene: Honoraria, Research Funding; Biomedis International: Honoraria, Research Funding; Mundipharma International: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Izutsu:Amgen: Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; MSD: Honoraria; HUYA Bioscience International: Research Funding; Celgene: Consultancy, Research Funding; Celltrion: Research Funding; Zenyaku: Research Funding; Sanofi: Research Funding; Gilead Sciences: Honoraria; Eisai: Honoraria, Research Funding; Novartis: Honoraria; Ono: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Nihon Medi-Physics: Honoraria; Symbio: Research Funding; Solasia: Research Funding; Mundhi: Honoraria; Otsuka: Honoraria; Bristol- Myers Squibb: Honoraria; Janssen: Honoraria, Research Funding; Astellas: Honoraria, Research Funding; Meiji Seika: Honoraria; Shionogi: Honoraria; Asahi Kasei: Honoraria. Tobinai:Takeda: Honoraria, Research Funding; SERVIER: Research Funding; Abbvie: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; HUYA Bioscience International: Consultancy, Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Chugai Pharma: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Eisai: Honoraria, Research Funding. Kobayashi:Pfizer: Research Funding; Ohtuka: Research Funding; Astellas: Research Funding.

Abstract: Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract: Background: The inhibition of BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has markedly improved the prognosis of chronic myeloid leukemia (CML). Recently, it has been recognized that some CML patients with a complete molecular response (CMR) are able to maintain treatment-free remission (TFR) after discontinuation of TKIs. However, no predictive prognostic factors for successful discontinuation of the treatment have yet been identified. We set out to further clarify the role of predictive biomarkers in molecular relapse and non-relapse after ABL TKI discontinuation. Materials and methods: Patients in sustained CMR (MR 4.5) undergoing TKI therapy were eligible for inclusion in the study. Molecular relapse was defined as loss of major molecular response (MMR) of at least one point. Genomic DNA was obtained from whole blood using a DNA Extractor WB Kit (Wako, Osaka, Japan), and was subjected to polymerase chain reaction (PCR) amplification using primers designed to detect a deletion site (2903 bp) in intron two of the BCL2L11 gene (forward: 5′-AATACCACAGAGGCCCACAG-3′; reverse: 5′-GCCTGAAGGTGCTGAGAAAG-3′) and JumpStart RedAccuTaq LA DNA polymerase (Sigma Aldrich, St. Louis, MO, USA). Results: 32 CML patients (17 men, 15 women, median age 58.4 years) were included in this study (Sokal category; low 24, intermediate 7, high 1). Six patients were treated with IFNα before TKI treatment, and 3 were treated with IFNα after stopping TKI. Median duration from TKI initiation to discontinuation was 79.3 months (range; 22 to 138 months); median duration of CMR before TKI discontinuation was 47.3 months (range; 5 to 97 months). Seven patients showed loss of MMR; 6 relapsed within 6 months and one showed late relapse at 25 months after discontinuation. The cumulative incidence of MMR loss was estimated as 18.8% at 12 months and at 24 months. Fluctuation of BCR-ABL transcript levels below the MMR threshold ( 〉 two consecutive positive values) was observed in 6.25% of patients at 24 months after ABL TKI discontinuation. Treatment-free remission was estimated as 81.2% at 12 months and at 24 months. The median period of restoration of second CMR was 6.0 months in re-treated patients. No patient died during the follow-up period. TKI-free remission was estimated as 78.1% at 30 months. There was only a significant difference in BCL2L11 (BIM) deletion polymorphism between the patients who maintained and those who lost MMR (p = 0.0253). No significant difference was observed in prior IFNα therapy, time to complete cytogenetic response (CCyR), time to MMR, and time to CMR between relapsing and non-relapsing patients. Conclusion: Our study shows a specific association between BCL2L11 (BIM) deletion polymorphism and clinical outcome after ABL TKI discontinuation in patients with long-lasting molecular undetectable residual disease. BCL2L11 (BIM) deletion polymorphism may predict relapse after ABL TKI discontinuation, which may have an impact on future ABL TKI discontinuation trials. These results further illustrate the importance of single nucleotide polymorphisms in successful long-term treatment of CML. Disclosures Ohyashiki: Bristol-Myers Squibb KK : Research Funding, Speakers Bureau; Novartis KK: Research Funding, Speakers Bureau.

Abstract: We analyzed 23 patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) showing der(1;7)(q10;p10) (hereafter der(1;7)), to identify the exact predictive factor of this cytogenetic change. Eight (34.8%) patients, including 6 MDS and 2 AML patients, had a prior history of genotoxic exposure, especially radiation and/or antimetabolites. Patients with der(1;7) consisted of three groups; one-third of patients had a prior history of genotoxic agents, one-third had additional cytogenetic changes at the time of MDS/AML diagnosis without prior exposure history, and the remaining one-third had neither prior exposure history nor additional cytogenetic changes. The current study demonstrated that the poor outcome of MDS/AML with der(1;7) is due to the high frequency of associated risk factors, i.e., prior history of genotoxic exposure, the presence of additional cytogenetic changes, or both. Identification of prognostic disadvantage might be required for appropriate strategy in managing MDS/AML patients with rare der(1;7) abnormality.

Abstract: Multiple myeloma (MM) is one of the common hematological malignancies and is a uniformly fatal disorder of B cells characterized by accumulation of abnormal plasma cells in the bone marrow.Clinical progression of patients with MM is improved with the proteasome inhibitor (PI) (e.g. bortezomib) and the immunomodulatory drugs (IMiDs) such as thalidomide and lenalidomide. Although PI and IMiDs have considerably changed the treatment paradigm of MM, many patients show disease relapse due to developing into drug resistance of MM cells. Since the prognosis remains poor for patients with refractory disease, the new therapeutic strategies are required to treat against these patients. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid. Two isoforms of sphingosine kinases (SphKs), SK1 and SK2, catalyze the formation of the S1P in mammalian cells. SphKs have also been shown to be up-regulated in the variety of cancer types. SphKs/S1P/S1P receptor (S1PR) axis is involved in multiple biological processes. It has been reported that S1P is involved in cell proliferation, angiogenesis and inflammation. S1P is also involved in cancer progression including cell transformation, oncogenesis and cell survival in hematological malignancies such as multiple myeloma. Therefore, S1P and SphKs may present attractive targets for MM treatment. One of the S1P analog, fingolimod (FTY720), which is an orally active immunomodulatory drug, is developed for the treatment of multiple sclerosis. SKI-I, which is a non-lipid pan-SphK inhibitor and ABC294640, selective inhibitor of SK2, are currently investigated in a pivotal phase 1 clinical trial against solid tumors. In this study, we investigated the efficacy of fingolimod, SKI-I, and ABC294640 by using the MM cell lines, RPMI8226, MM1.S and MM1.R. 72 hours treatment of fingolimod exhibited cell growth inhibition of MM cell lines in a dose dependent manner. Treatment of SKI-I and ABC294640 also exhibited cell growth inhibition in a dose dependent manner. Since S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis and chemotaxis, we next evaluated the chemotactic response of human umbilical vein endothelial cells (HUVEC). We found that 4 hours treatment of S1P significantly induced the migration of HUVECs compared to control medium. Treatment of HUVECs with fingolimod inhibited S1P-stimulated chemotaxis in a dose dependent manner. We also found that S1P-induced chemotaxis was abolished by the SKI-I and ABC294640. These results suggest that intracellular SK1 and SK2 may play the important role in S1P induced chemotaxis of HUVEC. We next investigated the S1P concentrations in MM patient by enzyme-linked immune sorbent assay (ELISA), because S1P is a potent tumorigenic growth factor that is likely released from tumor cells. We found that serum concentrations of S1P were significantly higher in patient with MM compared with normal samples. The average S1P levels of MM and normal control are 1503.431 and 1103.38 (p 〈 0.05). We also found that conditioned medium from MM cell line had chemotactic activity for HUVECs. These results implicate that S1P may be a novel biomarker for early stage of MM and that S1P is an important bioactive sphingolipid involved in angiogenesis. In this study, we also demonstrate that fingolimod, SKI-I and ABC294640 have potent preclinical anti-tumor activity in MM. These agents possibly inhibit angiogenesis with relation to MM cell growth and offer unique opportunities for novel therapeutic strategies for the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.