Kan Research Institute Research Articles

TAL1 Super Enhancer Aberration and Stil-TAL1 Fusion in Pediatric T Cell Acute Lymphoblastic Leukemia

Abstract Background Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer accounting for 10 to 15% of newly diagnosed pediatric ALL cases, and is associated with a poor treatment outcome compared with B-cell ALL especially in relapsed cases despite recent improvement with an intensive treatment. TAL1 deletion on chromosome 1p33 which is also known as STIL-TAL1, is found in 20-25% of cases with T-ALL, which results in overexpression of TAL1 under the control of the promoter of STIL located next to TAL1. Recently, TAL1 super enhancer aberration (TAL1-SE) was reported, which introduce binding motifs for the MYB transcription factor in a noncoding region. This somatic mutation results in aberrant expression of TAL1 without STIL-TAL1. Clinical features of TAL1-SE and its difference with STIL-TAL1 cases have been poorly studied, and this could be a new instrument of treatment stratification in T-ALL. Methods Targeted capture sequencing for coding regions of 151 genes using a custom-made bait including the TAL1-SE region was performed in 132 Japanese T-ALL cases under 15 years old to detect somatic mutations. For the further analysis, whole transcriptome sequencing (WTS) was performed in 88 cases. Screening for STIL-TAL1 fusion by reverse transcription PCR was also performed in additional 44 cases. All cases of TAL1-SE and STIL-TAL1 were validated by Sanger sequencing. Our pipelines,"Genomon 2" algorithm, were used for detection of somatic mutations and fusions. Most samples of DNA and RNA were offered from Tokyo Children's Cancer Study Group (TCCSG), and available clinical data were mainly based on the TCCSG clinical study treated with BFM-based ALL treatment protocol. Results TAL1-SE and STIL-TAL1 were found mutually exclusive in 6 (4.5%) and 24 cases (18.1%), respectively. All patients with TAL1-SE were under 10 years old. TAL1-SE cases showed a favorable 3-year overall survival (3y-OS) compared with STIL-TAL1 cases which were reported to show a good prognosis (83.3% vs 85.9% respectively, p = 0.71). However, 3-year disease free survival (3y-DFS) was 22.2% and 72.3% respectively (p = 0.04), indicating higher relapse rate in TAL1-SE cases, and they were probably rescued by second line treatment. Patients with STIL-TAL1 showed significantly poorer prednisolone response (over 1,000 /µl of blast at day 8, p < 0.05), though they showed favorable 3y-OS, which might be resulted from frequent high WBC counts at diagnosis (> 100×106/l, 59%). Comparison of TAL1 expression using read counts of WTS revealed significantly higher TAL1 expression in TAL1-SE cases than STIL-TAL1 cases (p < 0.05). To reveal the difference of gene expression profile between STIL-TAL1 and TAL1-SE, unsupervised consensus clustering of gene expression data in 24 cases performed WTS (6 cases of TAL1-SE and 18 cases of STIL-TAL1) divided into 2 clusters. All cases with TAL1-SE were grouped into the same cluster with 10 STIL-TAL1 cases (Cluster 1). The other 8 STIL-TAL1 cases were classified as Cluster 2. A genetic landscape of these 24 cases showed more frequent NOTCH1 and/or FBXW7 mutations in Cluster 1 (87.5% vs 37.5%, p = 0.02), whereas mutations in genes encoding transcription factor such as RUNX1 and BCL11B were found only in Cluster 1 (n = 1, n = 3, respectively). There was a tendency of better 3y-OS and 3y-DFS in Cluster 2 (73.9% vs 100%, p = 0.14, 55.6% vs 85.7%, p = 0.15, respectively). Conclusion TAL1-SE and STIL-TAL1 showed a relatively good prognosis, though high relapse rate in TAL1-SE and a poor prednisolone response in STIL-TAL1 were observed. STIL-TAL1 cases were biologically divided into 2 groups based on the expression profile (with TAL1-SE and STIL-TAL1 only), and these 2 groups showed different clinical and genetic features such as 3y-OS or frequency of NOTCH1/FBXW7 mutations. Our findings illustrate the clinicopathological differences between STIL-TAL1 and TAL1-SE cases, and thus, it might be helpful for development a new therapeutic strategy for these patients with TAL1 overexpression T-ALL. Disclosures Kataoka: Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Identification of a Novel Chromosomal Translocation t(11;16)(q23;q22) Fusing MLL to Enhancer of mRNA Decapping (EDC)-4 in Smoldering Acute Myeloid Leukemia

Abstract Background: Translocations involving the MLL gene located on chromosome 11q23 are usually found in de novo acute myeloid leukemia (AML) and generally confer a dismal prognosis unless the AF9 gene is involved (Döhner et al., Blood 2010;115:453-74). MLL can be fused to multiple different genes, resulting in the large and growing "MLL recombinome" (Meyer et al., Leukemia 2013;27:2165-76). Thus far, only two genes encoding proteins that are part of the mRNA decapping protein complex (i.e., DCP1A, DCPS) have been described as rarely being fused to MLL. Here, we describe an AML with an indolent disease course arising from myelodysplastic syndrome (MDS) that disclosed a unique MLL fusion with another component of the mRNA decapping complex, i.e., EDC4. Patient and Methods: Briefly, a 55 year-old female patient presented with an MDS [timepoint (t) -1] that within 10 months progressed to an AML with 2% blood and 40% bone marrow myeloblasts (t0). The patient refused treatment beyond supportive care. Six months later, a marked blast expansion of 80% was detectable in the blood (t1). The patient received 5 cycles of decitabine (t2, cycle 5), followed by 3 months of hydroxyurea (t3). Samples were depleted from CD3+ cells via MACS; CD3+ cells served as germline control. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), and the SureSelect Human All Exon v5 kit (Agilent Technologies) was used for exome capturing from gDNA. Next generation sequencing was performed on an Illumina Hiseq 2500. The analyses were performed as previously described (Kataoka et al. Nature Genetics 2015;47:1304-15, Becker et al. Blood 2014;123:1883-6). NOD scid gamma mice were used as hosts for patient derived tumor xenografts (PDX). Results: Standard metaphase cytogenetics at the diagnosis of AML (t0) revealed a previously undescribed translocation involving the MLL gene, i.e., t(11;16)(q23;q22), as the sole cytogenetic abnormality. The unknown fusion partner on chromosome 16 was identified by RNA-sequencing as the EDC4 gene (a.k.a. Ge-1), which encodes a key scaffold protein of the mRNA decapping complex; the fusion was confirmed by PCR on cDNA. The translocation led to the in-frame fusion of MLL exon 13 to EDC4 exon 6 which was linked by 19 nucleotides from EDC4 intron 5. The predicted amino acid sequence of the linker was ALNTLLR. Further analyses including exome sequencing on the samples collected over the disease course demonstrated STAG2 as a potential founder mutation that was already present during the MDS (t-1) and persisted throughout the disease course at variant allele frequencies (VAFs) of approximately 45-50%. At the time of transformation to AML (t0), the MLL-EDC4 and two RAS mutations (KRAS p.G13D, NRAS p.G12C) were detectable. Towards the terminal phase (t3), the RAS mutations disappeared and a clone that acquired a mutation in the FLT3 tyrosine kinase domain (TKD; p.D835V; VAF 43%) expanded. Primary blasts from the patient engrafted in NOD scid gamma mice and established a stable PDX serial transplantation mouse model used for drug testing. Conclusions: This report provides the first demonstration of an MLL-EDC4 in-frame fusion, with potential cooperativity with a founder mutation in the STAG2 splicing factor gene during the transformation of MDS to AML and the additional acquisition of a FLT3-TKD mutation during disease progression. RNA sequencing proved to be a very feasible approach to identify novel fusion partners of known oncogenes such as MLL. Disclosures Becker: BMS: Honoraria; Novartis: Honoraria. Kataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Schüler:Oncotest GmbH: Employment. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid.

Integrated Molecular Analysis of Myelodysplastic Syndromes Using Whole Genome Sequencing

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of myeloid malignancies characterized by refractory cytopenias with marrow dysplasia, which frequently progress to acute myeloid leukemia (AML). Although poorly understood in the previous era, the molecular events that underlie the pathogenesis of MDS have been intensively studied using advanced genomics in the past decade and are now fully catalogued into an array of well-defined functional pathways. However, mostly obtained through exome/targeted-capture sequencing, our knowledge about these molecular events is largely confined to those of single nucleotide variations (SNVs) and short indels, as well as arm-level copy number lesions, mostly within the coding sequences. Alterations in the non-coding regions, particularly a diversity of structural variations, in MDS genomes remain to be investigated in most part, even though the relevance of such lesions has recently been unequivocally demonstrated for other cancer types through large-scale whole genome sequencing (WGS) studies. Unfortunately, however, only a small number of MDS samples have been fully analyzes and inspected for genetic alterations using WGS.Patients and MethodsIn the present study, we performed an integrated, unbiased molecular study of 60 MDS cases, using whole genome sequencing (WGS) in combination with exome and transcriptome sequencing as well as methylome analysis. Paired tumor/germline DNA were obtained from patients' bone marrow and buccal smear samples. Sequencing data were analyzed using novel in-house pipelines, which were tuned to optimize detection of complex structural variations (SVs) and abnormalities in non-coding sequences. For some patients, multiple longitudinal materials were obtained along with their clinical course.ResultsWGS identified SNVs across the entire genome with a mean of 5.7/Mb/genome with a clear predominance of age-related C to T transitions, followed by other signatures. The spectrum of major targets of somatic mutations successfully recapitulated the previously reported one in MDS, including those involving splicing factors (SRSF2, SF3B1, U2AF1, and ZRSR2), epigenetic regulators (DNMT3A, ASXL1, TET2, BCOR, and EZH2), transcription factors (RUNX1, ETV6, and CUX1), signal transducing molecules (NRAS, KRAS, FLT3, PTPN11, CBL), and other critical molecules (TP53, NPM1, and STAG2). Moreover, other somatic variants within the coding regions were also identified that had already been reported in other human cancers but not in MDS, such as NCOR2X, MUC6, and TIAM2. The analysis of SVs unexpectedly revealed the complexity of MDS genomes. Most of the MDS genomes analyzed had a heavy burden of SVs including tandem duplications, deletions, translocations, and inversions, with a mean of 7.2/genome, which was far more than expected from conventional cytogenetics and array-based karyotyping. Complex rearrangements were common, frequently converging into particular chromosomes, suggesting multiple genetic events at a single genetic insult. Known targets of SNVs and indels were often affected by SVs, which largely escaped from conventional exome and targeted-capture sequencing, including RUNX1, TET2, FHITand other genes, suggesting that conventional platforms may substantially underestimate the frequency of alterations for some genes. Concomitant transcriptome analysis allowed to correlated abnormal splicing with somatic intronic events otherwise undetectable. Furthermore, comprehensive analysis of genomic aberrations in longitudinal samples enabled us to delineate the clonal architecture of the cellular population in MDS and their dynamics during the AML progression or clonal changes caused by AZA treatment.ConclusionsIntegrated molecular analysis using WGS and other platforms revealed the complexity of MDS genomes previously unexpected and reveal novel genetic alterations. Our results should help to extend our knowledge about the genomic landscape of MDS and provide novel insights into the molecular pathogenesis and clonal dynamics of MDS. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria. Naoe:Pfizer Inc.: Research Funding; CMIC Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Amgen Astellas BioPharma K.K.: Honoraria; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Celgene K.K.: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma Inc.: Research Funding. Kiyoi:Celgene Corporation: Consultancy; MSD K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; AlexionpharmaLLC.: Research Funding. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Genetic Profile of Acute Erythroid Leukemia

IntroductionAcute erythroid leukemias (AELs) are infrequent acute leukemias characterized by a predominant erythroid population, dysplasticity and ring sideroblasts. The 2016 revision of the World Health Organization (WHO) Classification re-classified previous AELs as myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) not otherwise specified (non erythroid subtype, or pure erythroid type), according to blast percentage in all nucleated bone marrow cells. Recent studies have offered the evidence that genetic profiles are associated with pathophysiology and clinical outcomes in myeloid malignancies. However, characteristic mutation profiles of AEL and its genetic differences or similarities with other-types of AML and MDS have not been fully elucidated due to the lack of a sufficient number of samples, although frequent TP53 mutations have been reported (35-43.5%).MethodWe performed a comprehensive genetic study, in which paired tumor/normal DNA from 24 AEL cases were analyzed by whole-exome sequencing (WES). Subsequently, in total 41 cases were screened for mutations in 69 driver genes associated with myeloid malignancies using targeted-capture sequencing. Genome-wide copy number analysis was also performed based on allele frequency of heterozygous single nucleotide polymorphisms and sequencing depth. Frequencies of driver mutations in AEL were compared to those in other-types of AML analyzed by the Cancer Genome Atlas Network (TCGA) (N = 197).ResultsThe mean number of nonsynonymous mutations identified by WES was 10 per sample (range, 0-21), which was similar to that in other-types of AML or MDS. Most frequently mutated genes were STAG2, TP53, RUNX1 and TET2 (17%, respectively), followed by WT1 and BCOR (13%, respectively). All TP53 mutated cases harbored complex karyotypes. Subsequent targeted sequencing identified 3.4 mutations per sample on average (range, 0-9). TP53 was mutated in 8 cases (20%), and lower frequency of TP53 mutations in our cohort was attributed to the relatively lower proportion (21%) of cases with complex karyotype compared to those in the previous reports. Commonly mutated genes in other-types of AML, such as NPM1, FLT3, and DNMT3A, were relatively infrequent in AEL and affected 7 (17%), 4 (9.8%) and 3 (7.3%) cases, respectively. Genes associated with splicing machinery were mutated in 5 cases (12%), including SRSF2 (9.8%) and U2AF1 (2.4%). Of note, mutations in genes implicated in cohesin complex were observed in as many as 32% of cases, including those in STAG2 (24%), SMC1A (4.9%) and RAD21 (2.4%). Mutations in epigenetic regulators were also frequently identified in AEL, including those in WT1 (20%), TET2 (15%) and IDH1/2 (7%). Mutations in TP53 were mutually exclusive with those in cohesin complex (p = 0.04) and epigenetic regulators (p = 0.05). When we compared the frequencies of recurrent mutations in AEL to those in other-types of AML analyzed by TCGA, mutations in STAG2 (p = 6*105), SRSF2 (p = 7*104), WT1 (p = 0.01), GATA2 (p = 0.008), and BCOR (p = 0.02) were significantly more frequent in AEL, whereas those in FLT3 (p = 0.01) and DNMT3A (p = 0.007) were significantly less frequent in AEL. Mutations in cohesin complex and splicing factors were frequently observed both in AEL and MDS, and higher frequency of these genetic lesion in AEL rather than other-types of AML suggested that AEL and MDS are similar disease entities. Recently, genomic subgroups of AML with mutations in cohesin complex, spliceosome and TP53 have been associated with poor prognosis in AML, and poor prognosis of AEL was thought to be due to higher frequency of these genetic lesions.ConclusionWES and follow-up targeted sequencing revealed the genetic landscape of AEL. Frequent mutations in TP53, splicing factors and cohesin complex were characteristic to AEL, and thought to be involved in its pathophysiology. Our data showed the genetic similarities between AEL and MDS, which were compatible with the partial integration of AEL into MDS in the recent revision of WHO classifications. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Heuser:Pfizer: Research Funding; BerGenBio: Research Funding; Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; Celgene: Honoraria; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding.

Molecular Heterogeneity in Peripheral T-Cell Lymphoma Not Otherwise Specified Revealed By Comprehensive Mutational Profiling

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoproliferative disorders arising from mature T-cells. Among them, PTCL-not otherwise specified (PTCL-NOS) is a diagnosis of exclusion, comprising the largest fraction of PTCL with a diverse underlying pathogenesis. Recently, the concept of nodal T-cell lymphomas with T-follicular helper (TFH) phenotype, including angioimmunoblastic T-cell lymphoma (AITL) and PTCL-NOS that manifests a TFH phenotype, has been proposed, a distinguishing feature of which is the high frequency of TET2, IDH2, DNMT3A, and RHOA(G17V) mutations. Although recent large-scale genetic studies have uncovered mutational landscapes of several other subtypes of PTCLs, such as cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma (ATL), the entire picture of somatic alterations in PTCL-NOS still remains elusive. In addition, their similarities and differences among various histological subtypes in PTCLs have not been fully elucidated.To address this issue, we initially analyzed our and publicly available whole-exome/genome as well as transcriptome sequencing data from PTCL-NOS and other related PTCLs. Then, we carried out an extensive investigation of somatic mutations and structural variations (SVs) in PTCL-NOS using targeted-capture sequencing of 118 PTCL-NOS samples. Consistent with previous reports, TET2 (35%) was the most frequently mutated gene in PTCL-NOS with the majority (78%) affected by multiple mutations, followed by RHOA (25%), TP53 (16%), KMT2C (12%), PLCG1 (12%), and HLA-B (11%). Besides them, a considerable proportion of patients harbored mutations in components of T-cell receptor (TCR) /NF-κB pathway (such as PRKCB, CARD11, IRF4, and PRDM1), other signal transduction molecules (STAT3, NOTCH1, and SOCS1), chemokine receptors (CCR4 and CCR7), epigenetic modifiers (CREBBP, KDM6A, IDH2, and DNMT3A), transcriptional regulators (GATA3 and TBL1XR1), and molecules associated with immune evasion (HLA-A, HLA-B, FAS, B2M, and CD58). In addition to deteriorating SVs involving frequently affected genes (TP53, FAS, GATA3, and TBL1XR1), we discovered several genes almost exclusively affected by SVs, including TP73, IKZF2, and NFKB2, and CD274.Novel targets of recurrent mutation were also identified, including PDCD1, YTHDF2, and LRP1B, which were frequently targeted by nonsense and frameshift mutations distributed throughout the entire genes. Among them, PDCD1encodes PD-1 receptor transmitting an inhibitory signal from PD-L1 and PD-L2 ligands in T cells, and its loss of function seems to enable tumor cells to escape from the suppression by this negative signal. Although the roles of YTHDF2, a reader protein of N6-methyladenosine, and LRP1B, a member of the low density lipoprotein receptor family, in T cells are not immediately apparent, these findings shed light on a new biological function of these genes.Next, we investigated the co-existence relationship between frequently altered genes in PTCL-NOS. Interestingly, mutations characteristic of TFH lymphomas (TET2, RHOA, IDH2, and DNMT3A) tended to co-occur in a subset of PTCL-NOS cases, whereas they were almost mutually exclusive with mutations in TP53 and TCR/NF-κB pathway genes. This observation reveals the molecular distinction between TFH and non-TFH lymphomas in PTCL-NOS: the former is similar to AITL, although TET2 mutations did not show higher allelic burden than RHOA and IDH2mutations. In contrast, the latter is at least partly characterized by the genetic alterations shared with ATL.In summary, our findings illuminate the landscape of somatic alterations in PTCL-NOS and provide a novel insight into their genetic and molecular heterogeneity, which would help us to exploit a new therapeutic strategy to combat this disease. DisclosuresOhshima:CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria.

Landscape of Driver Mutations and Their Clinical Impacts in Pediatric Acute Lymphoblastic Leukemia

IntroductionB-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner.MethodsWe enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection.ResultsThe median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]).Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation.Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified.Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL.ConclusionsWe revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Comprehensive Genetic Analysis in Cases of Juvenile Myelomonocytic Leukemia for Prognostic Estimation

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm (MPN) that occurs during childhood and has a poor prognosis. Somatic or germline mutations in canonical RAS pathway genes, i.e., PTPN11, NF1, NRAS, KRAS, and CBL, are reported be detected in approximately 85% patients. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for JMML. Although spontaneous remission is occasionally observed in others with supportive therapy, some patients show aggressive disease progression despite HSCT. Recent studies have identified several additional genetic events in an array of genes, including SETBP1 and JAK3, but the relationship between genetic alterations and clinical outcomes remains unclear.Patients and Methods: A total of 131 patients (88 boys, 43 girls) with JMML were enrolled in the study. The median age was 15 months (range, 1-160 months). Eighty-two of 131 patients underwent HSCT, and 36 patients died (disease related, n = 27, transplantation-related complications, n = 16, infection, n = 5, unknown, n = 3). We performed comprehensive genetic analyses of the 131 JMML patients using whole-exome sequencing (n = 68, 52%) or targeted deep sequencing (n = 92, 70%), and assessed the impact of genetic alterations on clinical outcomes in 119 patients, excluding 12 patients with Noonan syndrome-related myeloproliferative disorder (NS/MPD).Results: We identified canonical RAS pathway gene mutations in 115 of 131 patients (88%). Although most RAS pathway mutations were mutually exclusive, coexisting secondary RAS pathway mutations were found in nine patients (8%). In addition, 28 patients harbored secondary genetic alterations in other genes, including SETBP1 (n = 10), JAK3 (n = 12), ASXL1 (n = 6), SH3BP1 (n = 1), RRAS2 (n = 2), and SOS1 (n = 3). In total, 34 of 131 patients harbored secondary genetic mutations. In univariate analysis, patients with secondary genetic mutations showed poorer survival rates than patients without these mutations [5-year transplantation-free survival (TFS) (95% CI) = 8.8% (2.3%-21.1%) vs. 24.1% (15.2%-34.1%), p = 0.007; 5-year overall survival (OS) (95% CI) = 49.6% (32.0%-65.0%) vs. 62.3% (50.8%-71.8%), p = 0.135]. On the basis of the dominant canonical RAS pathway mutations classification, patients with PTPN11 and NF1 mutations were significantly associated with the presence of secondary genetic mutations compared to patients with other RAS pathway gene mutations (PTPN11 (20 of 43, 47%), NF1 (5 of 7, 71%), NRAS (2 of 18, 11%), KRAS (4 of 20, 20%), CBL (1 of 17, 6%), p < 0.001). Consistent with previous reports, patients with PTPN11 and NF1 mutations had inferior survival rates than other JMML patients [5-year TFS (95% CI) = 0% vs. 32.7% (21.5%-44.3%), p < 0.001; 5-year OS (95% CI) = 45.3% (31.1%-58.5%) vs. 68.1% (55.2%-78.0%), p = 0.006]. Multivariate survival analysis identified the RAS pathway mutations (i.e., patients with PTPN11 and NF1 mutations vs. others) [TFS: HR (95% CI) = 3.732 (2.382-5.847), p < 0.001; OS: HR (95% CI) = 1.983 (1.117-3.521), p < 0.019] and low platelet count (<33 × 109/L vs. ≥ 33 × 109/L) [TFS: HR (95% CI) = 1.816 (1.160-2.843), p < 0.001] as independent risk factors for TFS and OS. In subgroup analysis of 50 patients with PTPN11 and NF1 mutations, there were no significant survival differences between patients with (n = 25) or without (n = 25) secondary genetic mutations [5-year TFS (95% CI) = 0% vs. 0%, p = 0.753; 5-year OS (95% CI) = 39.6% (20.8%-57.9%) vs. 51.7% (30.9%-69.1%), p = 0.589].Discussion: Consistent with previous studies, secondary genetic mutations were associated with inferior survival rates, but high correlations were observed in JMML patientswith PTPN11 and NF1 mutations. Our results suggest that comprehensive genetic mutational profiling is essential to estimate prognosis and to stratify JMML patients who require HSCT and/or novel treatment modalities. DisclosuresOgawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Kojima:SANOFI: Honoraria, Research Funding.

Genome-Wide Mutational Landscape of Infant Acute Lymphoblastic Leukemia

[Background] Mixed lineage leukemia (MLL also called KMT2A) rearrangement-positive leukemia is one of the most aggressive types of leukemia. It is diagnosed predominantly in infants and typically shows a multi-lineage phenotype. Since current chemotherapy fails in more than 50% of infantile acute lymphoblastic leukemia (ALL) with MLL-rearrangement, a better understanding of biological features of the disease is important in order to develop more specific and successful treatment strategies. Despite the evident biological and clinical significance of MLL-rearrangement, it has also been shown in vitro and in vivo that MLL-rearrangement is not sufficient to induce full leukemic transformation, indicating additional hits are required for complete leukemogenesis.Recently, sequencing-based genome-wide studies have suggested collaborative involvement of RAS-PI3K pathway in infant leukemogenesis through identification of recurrent mutations affecting RAS-PI3K pathway genes, in infant ALL with or without MLL-rearrangement. However, genetic basis of infant ALL with MLL-rearrangement are not fully elucidated.[Materials and Methods] Here, we performed whole exome sequencing in leukemia samples with matched peripheral blood T lymphocyte from 46 cases of infant ALL including 3 cases with germline MLL. Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. For mapping and mutation calling, we used publicly available "Genomon Pipeline".[Results] In total, 198 somatic non-silent mutations (4.3 mutations per case) were identified, including 125 missense, 4 nonsense, 8 splice site mutations, 49 frameshift indels, and 12 in-frame indels. Thirteen genes were recurrently affected, among which RAS-PI3K pathway mutations were the most frequent: KRAS (10/46 cases, 21.7%), NRAS (4/46 cases, 8.7%) and PTPN11 (3/46 cases, 6.5%). In addition, novel recurrent mutations were detected in HSP90AB1 (5/46 cases, 10.9%), MAPRE3 (2/46 cases, 4.3%) and SRCAP (2/46 cases, 4.3%), as well as known candidate driver mutations in FLT3 (7/46 cases, 15.2%), TP53 (3/46 cases, 6.5%), CHD4 (2/46 cases, 4.3%) and PAX5 (2/46 cases, 4.3%). HSP90AB1 is a member of Heat shock protein90 (Hsp90) family that encodes a group of molecular chaperones involved in stabilization and activation of multiple oncogenic proteins and pathways. Overexpression of Hsp90 is observed in ALL, chronic myeloid/lymphocytic leukemia and several non-hematological malignancies. Another recurrently mutated gene, SRCAP, encodes the core component of chromatin-remodeling Snf2-related CREBBP activator protein complex. Recurrent mutations in this gene have recently been reported in FLT3-ITD positive acute myeloid leukemia with significantly high co-occurrence of MLL3 mutations. Overall, RAS-PI3K pathway mutations were detected in 19/46 cases of infant ALL (41.3%). In our cohort, none of 3 germline MLL cases contained RAS-PI3K pathway mutations; one case had a PAX5 mutation, another had a SRCAP mutation, and the other had no candidate driver mutations. In the germline MLL case without candidate drivers, G-banding had identified a structural abnormality, t(5;15)(p15;q11.2), that is rare but reported to be predominant in infant ALL rather than other hematological/non-hematological malignancies.[Conclusion] Our results demonstrated that aberrations of cell proliferation signaling, transcription factors, as well as cell cycle/epigenetic regulators are co-operative oncogenic events in MLL-rearrangement cases. The mutation spectrum is similar to myeloid malignancies rather than lymphoid cancers. Intriguingly, we identified novel recurrent mutations in the oncogenic proteins, such as HSP90AB1, MAPRE3, and SRCAP,suggesting that these are potentially targetable by small-molecule therapy. In addition, we also illustrated the genetic differences between MLL-rearrangement and germline MLL. Thus, our mutational landscape provides a novel insight into the molecular mechanisms of infant ALL and may contribute to improving the clinical outcome of infant patients suffering from the intractable ALL. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Regeneration of Tumor Antigen Specific CTLs Utilizing iPSC Technology for Off-the-Shelf Immunotherapy

In the tumor immunotherapy field tumor antigen specific T cell therapy is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen specific T cells without exhaustion. To solve this problem, we are now utilizing induced pluripotent stem cell (iPSC) technology. When iPSCs are established from tumor antigen specific cytotoxic T cells (CTLs), T cells regenerated from these iPSCs (T-iPSCs) are expected to express the same T cell receptors (TCRs) as the original CTLs. Therefore, it will become possible to obtain de novo generated tumor antigen specific CTLs almost unlimitedly. In line with this concept, we have succeeded in establishing iPSCs from CTLs specific for the melanoma antigen MART-1 and regenerating MART-1 specific CTLs (Vizcardo et al, Cell Stem Cell, 2013).The strategy mentioned above was supposed to be applied as autologous transfusion. Autologous transfusion setting, however, has some problems; i) high cost to establish T-iPSCs from each patient, ii) heterogeneity of the quality as iPSCs, iii) difficulty in getting T-iPSCs with high affinity TCRs. To solve these problems, we are now planning to apply this method to the allogeneic transfusion setting. In allogeneic setting, we can select high quality T-iPSC clones with high affinity TCRs and such clones can be utilized to many patients with the same HLA type. In this context, it is preferable to establish T-iPSCs from HLA haplotype homozygous donors, since CTLs generated from these T-iPSCs can be administered to the patients carrying the same HLA haplotype at least on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured.Now we are trying to establish T-iPSCs from CTLs specific for various tumor antigen from healthy donors. We succeeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific cytotoxicity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to lyse some leukemia cell lines and primary leukemia cells which express endogenous WT1 protein. Administration of regenerated CTLs prolonged the survival of mice inoculated with leukemia cell line in in vivo xenograft model. In addition, we succeeded in transducing iPSCs derived from non-T cells with WT1-specific TCR genes (TCR-iPSCs) and generating CTLs from them. Generated CTLs from TCR-iPSCs exhibited comparable TCR specific cytotoxicity as regenerated CTLs from T-iPSCs. Using this method, it would be much easier to produce CTLs with defined high affinity TCRs specific for various tumor antigen.We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPSCs/TCR-iPSCs from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS/TCR-iPSCs cell bank", with the aim of making off- the-shelf tumor immunotherapy a reality. DisclosuresKataoka:Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kawamoto:Regcel: Research Funding.

Predictive Value of Mutation Analysis in the Diagnostic Approach to Patients with Unexplained Cytopenia

▪The diagnostic approach to unexplained cytopenia is hampered by the poor specificity of dysplastic changes that may complicate the distinction between myeloid neoplasms (MN) and non-malignant cytopenias. In the last years, several somatic mutations were identified in MN; however, the diagnostic value of mutation analysis needs to be defined. In this study, we performed a mutation screening in a prospective cohort of patients with unexplained cytopenia undergoing a comprehensive diagnostic work-up, with the aim to estimate the predictive value of somatic mutations.This study included two cohorts: a learning cohort that consisted of 683 consecutive patients investigated for unexplained cytopenia at the Policlinico San Matteo & University of Pavia, Italy, between 2003 and 2015; and a validation cohort, including 190 patients referred as second opinion for suspected MDS. A set of 42 genes was analyzed on DNA from peripheral blood granulocytes using Illumina HiSeq (Illumina Inc., CA, USA). The diagnosis of patients in the learning cohort was MN in 409 cases (233 MDS, 86 MDS/MPN, 35 MPN; 55 AML), other cytopenia in 120 cases, whereas in 154 patients a provisional diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) was adopted. After a median follow-up of 22 months (range 3-136), 38 patients in this category developed a MN (ICUS-MN).The most frequently mutated genes were TET2 (171/683, 25%), ASXL1 (15%), SRSF2 (14%), SF3B1 (11%), DNMT3A (10%), RUNX1 (9%). Significantly higher number of mutations per subject and variant allele frequency (VAF) were observed in MN (n=2, range 0-9; VAF=0.39, 0.03-0.57) compared with ICUS (n=0, 0-7; VAF=0.31, 0.03-0.51) or other cytopenia (n=0, 0-2; VAF=0.06, 0.03-0.44) (P<0.001). Fifty-seven of 409 (14%) MN patients did not carry any mutation in the set of analyzed genes. Among these, 6 had MDS del(5q), while 18 received a diagnosis of MDS based on mild dysplasia with normal karyotype, and showed a 5-year probability of progression of 0%, suggesting that a diagnosis of MDS might have been not appropriate despite WHO criteria. When accounting for these cases, an unmutated status had a negative predictive value for MN of 0.83. Conversely, having two or more mutations had a positive predictive value (PPV) for MN of 0.94. We then performed ROC analyses to explore the cut-off value of VAF with the highest predictive value for MN, and found that a VAF equal or higher than 0.10 had a PPV for having or developing MN of 0.92.Next, we calculated the predictive value for diagnosis of MN of the most frequently mutated genes. In multivariable logistic regression, having two or more mutations (OR=4.07, P<0.001) or carrying SF3B1 mutation (OR=3.56, P=0.016) were independent predictors for MDS or other MN. SF3B1 mutation showed a specificity for myelodysplasia of 0.99, suggesting that this lesion may provide presumptive evidence of MDS even in the absence of definitive morphological features. RNA splicing genes (SF3B1, SRSF2, U2AF1) and RUNX1 had the highest predictive value for MN irrespective of co-occurring mutations, PPVs ranging from 0.88 to 0.93. PPVs increased to 0.97-0.99 when accounting for ICUS-MN as true positive cases. Conversely, PPVs of mutations in genes of DNA methylation (TET2, DNMT3A) and chromatin (ASXL1) as isolated lesions ranged from 0.39 to 0.69, while having one or more co-mutated genes significantly increased PPVs to 0.88-0.96.Among patients with a diagnosis of ICUS, 57 of 154 (37%) carried one or more mutations (Clonal Cytopenia of Undetermined Significance, CCUS). Patients with CCUS showed a significantly higher probability of developing MDS compared with those without evidence of clonality (HR=7.48, 10-year cumulative probabilities of progression: 96% vs 15% respectively, P<.001).Finally, the predictive values of mutation analysis were tested in the independent validation cohort of patients referred to our institution for suspected MDS, and were fully confirmed.In conclusion, selected mutated genes or co-mutation patterns may identify patients with high likelihood of having MDS or other MN. The definition of a category of CCUS allows to recognize with high sensitivity patients who do not fulfill diagnostic criteria but are at high risk of developing MDS. Taken together, these data suggest that mutation analysis on peripheral blood cells may significantly improve current diagnostic approach to patients with unexplained cytopenia. DisclosuresOgawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding.

Recurrent VAV1 Abnormalities in Angioimmunoblastic T Cell Lymphoma

Background: Angioimmunoblastic T cell lymphoma (AITL) is a subset of peripheral T cell lymphomas (PTCLs). AITL has very specific clinical features, including high fever, skin rush, and autoimmune-like manifestations. The molecular pathogenesis of AITL is poorly understood despite the recent progress in genetics of this disease. We and others previously identified disease specific ras homolog family member A (RHOA) mutations together with muations in epigenetic regulators, tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3 alpha (DNMT3A) mutations, and isocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2) in AITL. Thesemutations were also frequent in PTCL, not otherwise specified (PTCL-NOS) having features of AITL.RHOA, a small GTPase is predominantly activated by guanine nucleotide exchange factors (GEFs). VAV1encodes a GEF, serving as an important mediator of T-cell receptor signaling pathway. Phosphorylation of VAV1 occurs within seconds in response to antigen stimulation of the T-cell receptors by Syk and Src-family tyrosine kinases and initiate downstream signaling.Objective: We aim to identify novel disease specific gene mutations in AITL besides RHOA.Methods: We performed RNA sequencing of 9 PTCL samples, including 6 AITL and 3 PTCL-NOS. Targeted deep sequencing of VAV1 was performed for 139 PTCL samples, including 93 AITL and 46 PTCL-NOS, 48 of which have RHOAmutations.VAV1 wild-type (WT), VAV1-STAP2, and VAV1 mutant (p.173_177del, p.165_174del, and p.Pro615Leu) cDNA was subcloned into pEF vector. Nuclear factor of activated T cell (NFAT) activity in response to CD3 stimulation was examined in Jurkat cells transiently transfected with a reporter vector containing NFAT response element (NFAT-RE) together with VAV1 WT and mutant cDNAs. The levels of interleukin-2 (IL-2) in response to CD3 stimulation were examined for the supernatant of Jurkat cells inducibly expressing VAV1 WT or VAV1-STAP2 cDNAs. Rac1 activation was examined in NIH3T3 cells transiently transfected with VAV1WT and mutant cDNAs.Results: RNA sequencing identified a fusion gene involving VAV1 and STAP2 in an AITL sample without RHOA mutations. Moreover, targeted sequencing of VAV1 identified 2 in-flame deletion mutations in an acidic region (c.C518_529del:p.173_177del and c.C494_520del:p.165_174del) in AITL samples and 2 missense mutations in a zinc finger and SH3-SH2-SH3 module (c.G1668C:p.Glu556Asp and c.C1844T:p.Pro615Leu) in PTCL-NOS and AITL samples, respectively. All of these VAV1 mutations were found in the samples without RHOA mutations. The phosphorylation of VAV1 at Tyr 174 was enhanced in Jurkat cells expressing VAV1-STAP2 cDNA than those with VAV1 WT cDNA or mock. The phosphorylation was efficiently blocked by Src inhibitors, PP2 and dasatinib. Transient transduction of VAV1-STAP2, VAV1 p.173_177del, p.165_174del, and p.Pro615Leu cDNA in Jurkat cells resulted in enhanced NFAT activity. Moreover, the aberrant reporter activity was blocked by Src-family kinase inhibitors. Jurkat cells inducibly expressing VAV1-STAP2 cDNA showed higher IL-2 secretion than those with mock or VAV1 WT. RAC1 activation was higher in NIH3T3 cells transfected with VAV1 p.173_177del and p.165_174del mutant cDNA than those with VAV1 p.Pro615Leu mutant and VAV1-STAP2 cDNA.Conclusions: Recurrent VAV1 abnormalities were found in AITL and PTCL-NOS samples. Our data suggest that the VAV1 mutations may contribute to its clinical features and the VAV1 mutants can be a new therapeutic target. DisclosuresIzutsu:Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Structural Variations Involving Programmed Death Ligands in B-Cell and T-Cell Lymphomas

Immune checkpoint blockade using anti-PD-1 or anti-PD-L1 antibodies is a highly promising therapy that can induce a durable anti-tumor response and a long-term remission in many patients with multiple cancer types. In particular, the excellent efficacy of anti-PD-1 antibody has been reported in advanced cases with classical Hodgkin lymphoma (cHL), of which high frequency of genetic lesions involving PD-L1 and/or PD-L2 somatic alterations is a defining feature, suggesting a close link between the relevant genetic lesions and the efficacy of anti-PD-1/PD-L1 therapy. In addition to cHL, several subtypes of B-cell lymphomas are shown to have structural variations (SVs) involving PD-1 ligands, such as gene amplification and chromosomal translocation causing promoter replacement. Moreover, recently we reported unique SVs disrupting the 3′-untranslated region (UTR) of PD-L1 in a diversity of cancers, including adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma (DLBCL). However, the comprehensive landscape of PD-L1 and PD-L2 alterations in non-Hodgkin lymphomas has not been fully elucidated. Therefore, in this study, we interrogated PD-L1 and PD-L2 genetic aberrations and characterized their features in a variety of non-Hodgkin lymphomas.To do this, lymphoma-derived DNA was captured for the entire region of PD-L1 and PD-L2 genes including their exons, introns, and 3′- and 5′-untranslated regions (UTRs) and subjected to high-throughput DNA sequencing. More than 300 samples from different lymphoma subtypes were analyzed, including DLBCL, follicular lymphoma, mantle cell lymphoma, MALT lymphoma, primary mediastinal B-cell lymphoma, peripheral T-cell lymphoma-not otherwise specified, and cutaneous T-cell lymphoma. We also analyzed publicly available sequencing data as well as our own data for lymphomas, which included Burkitt and angioimmunoblastic T-cell lymphomas as well.PD-L1/PD-L2-involving SVs were most frequently observed in PMBCL, accounting for 26.3% of the cases, but widely observed in various B- and T-cell lymphomas at varying but generally low frequencies. However, in contrast to PD-L1-involving SVs, which were found in both B- and T-cell lymphomas, PD-L2-involving SVs were exclusively seen in B-cell lymphomas. Depending on samples, different SV types were observed, including deletion, inversion, tandem duplication, and translocation, but most of SVs resulted in a truncation of the 3'-UTR of the PD-L1 or PD-L2 genes. Unlike previous reports, we rarely found those SVs that translocate PD-L1/PD-L2 to an ectopic regulatory element. Of particular interest were those cases in which multiple, independent SVs that converged to PD-L1 and PD-L2, were observed in a single tumor sample, underscoring the importance of PD-L1 and PD-L2 SVs in clonal selection and expansion of these tumorsGiven that PD-L1-involving SVs are detected not only in aggressive lymphomas but also in a variety of solid cancers, we hypothesized that PD-L2 genetic alterations are also present in other human cancers. However, no PD-L2-involving SVs were identified among > 10,000 cancer samples from 32 tumor panels, for which RNA sequencing data were available from the Cancer Genome Atlas (TCGA). These results suggest that PD-L1 is affected in a broad spectrum of human malignancies, whereas PD-L2 SVs are a characteristic alteration of B-cell lymphomas, which is consistent with their expression patterns.Based on these findings, we assessed whether disruption of PD-L2 3'-UTR also induces PD-L2 overexpression as seen for that of PD-L1 3'-UTR. When introduced in T2 human B and T lymphoblast hybrid cell line using the CRISPR/Cas9 system, SVs involving an almost entire PD-L2 3'-UTR sequence actually induced a significant elevation of PD-L2 expression, confirming the relevance of 3'-UTR in the regulation of PD-L2 expression.Taken together, our findings clarified the entire picture of PD-L1/PD-L2-involving SVs ligands in B- and T-cell lymphomas. Detection of these SVs might help the identification of patients with non-Hodgkin lymphomas who potentially benefit from PD-1/PD-L1 blockade therapy. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria. Izutsu:Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding. Ohshima:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

VEGFA- a New Therapeutic Target in CNS Leukemia

Acute lymphoblastic leukemia (ALL) is the commonest cancer to affect children. Historically, central nervous system (CNS) involvement has been considered as a feature of high risk disease. Although this risk is mitigated by current intended CNS directed treatments based on risk stratification strategies, currently 10% or more of children will die from relapsing or unresponsive disease and the CNS is still a major site of relapse.CNS directed treatments bypass the blood brain barrier (BBB) but do not specifically target CNS leukemic cells. Consequently, although effective, they are associated both with serious short-term toxicities and debilitating long-term morbidities. Understanding the biology of those ALL cells which lodge in the CNS may lead to the development of drugs to specifically target them, and this could further improve outcomes but with fewer complications.We transplanted bone marrow (BM) derived leukemic cells from children with B-ALL into non-conditioned immune-deficient mice by tail-vein injection. We confirmed BM engraftment and evidence for CNS involvement was demonstrated by microscopy and flow cytometry analyses of the CSF, and was confirmed histo-pathologically in brain tissue. The pattern of leukemic infiltration of the CNS in xenograft recipients closely mimicked that seen post mortem in children with ALL. Furthermore, the MRI scans of xenograft mice showed a meningeal enhancement typical of the leukemic meningitis seen in patients with CNS involvement. Thus by using standard clinical techniques, we were able to demonstrate that the xenograft recipients of B-ALL cells faithfully recapitulates patterns of CNS disease that are seen in human.Interestingly, human leukemic cells isolated from either the CNS or BM of individual recipient mice demonstrated different kinetics of engraftment following serial passage into secondary and quaternary recipients. This functional observation implies cell intrinsic differences between leukemic cells lodged at the two sites.We next compared the gene expression profiles of leukemic cells isolated from the CNS and BM of affected children and xenograft recipients. Using gene set enrichment analysis, we demonstrate that leukemic cells that have infiltrated the CNS, in contrast to those in the BM, show features of adaptation to hypoxia including cell cycle quiescence and downregulation of oxidative phosphorylation. Consistent with these transcriptional data, we could demonstrated that fewer of the CNS-derived leukemic cells were in S/G2/M compared with BM-derived leukemic cells and that CNS-derived leukemic cells consumed less oxygen in in vitro assays.We found that CNS derived leukemic cells had a transcriptional signatures suggestive of physiological adaptation to hypoxic microenvironment and VEGFA is one of the most up-regulated genes in CNS-derived leukemic cells. We tested therapeutic potential of targeting VEGFA using bevacizumab, a VEGFA-neutralizing antibody. Bevacizumab or normal saline (NS) was injected intraperitoneally into the xenografted recipients of B-ALL cells. In the bevacizumab group, leukemic cell numbers in the CNS were significantly less at the end of treatment, and staining of brain sections confirmed reduced leukemic burden in CNS of bevacizumab-treated mice compared with control mice. This in vivo preclinical assay shows that VEGFA could be targeted to treat the patients with ALL who have CNS involvement.In conclusion, we have shown that B-ALL cells residing in the CNS have gene expression signatures and phenotypic features that are distinct from those in the BM, and that this can be effectively targeted in preclinical model of CNS leukemia. These observations may inform not only further mechanistic research, but also future clinical trials. DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia

Pediatric ALL is the most common childhood tumor and the leading cause of childhood cancer deaths. To gain a better understanding of the landscape of somatic mutations in ALL, we performed whole exome and targeted sequencing of 240 pediatric B-ALL patients with their matched remission samples. The significantly mutated genes fall into several common categories: RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment and p53/cell cycle pathway.RAS/receptor tyrosine kinases: the most frequently mutated genes were members of RAS signaling (NRAS, KRAS, FLT3, PTPN11). Besides the well know hotspot mutations [G12D/V/C (NRAS 13 cases, KRAS 13 cases), G13D (NRAS 14 cases, KRAS 11 cases) and Q61H/L/R/K (NRAS 15 cases, KRAS 1 case)], novel mutational sites were also identified for KRAS: A146T/P (3 cases), K117N/T (4 cases) and V14I (1 case). High frequency missense mutations of PTPN11 clustered in SH2 domain (included the canonical hotspot A72T (5 cases) and E76K/V (4 cases)) and tyrosine-phosphatase catalytic domain (G503R/V). For FLT3, well-appreciated activating hotspot mutations in the kinase domain (D835Y/Y842C) and several novel recurrent mutationswere identified.Epigenetic regulators: hotspot mutations were identified in histone H3K36 methyltransferase WHSC1. Mutation E1099K located in the SET domain, was identified in 10 patients as well as two of the 5 ALL cell lines that we sequenced (RS4;11, SEM). Stable silencing of E1099K mutant WHSC1 in RS4;11 cells by either lentiviral shRNA or CRISPR guide RNA (sgRNA) markedly reduced clonogenic growth both in vitro and in vivo, underscoring the critical role of WHSC1 in lymphoid malignancies. Two highly-related histone/non-histone acetyltransferases, CREBBP and EP300, were also prominently mutated in our cohort. Mutations of CREBBP predominantly occurred in the acetyltransferase domain, particularly in the hotspot R1446C/H. Mutations of chromatin remodeling genes (ARID1A and ARID2) have been identified in a number of cases. Silencing of ARID1A in ALL cell lines by lentiviral shRNA resulted in upregulation of the pro-growth regulator c-MYC, while forced expression of ARID1A reduced c-MYC luciferase reporter activity. In addition, silencing of ARID1A by either shRNA or CRISPR-sgRNA resulted in enhanced clonogenic growth, suggesting that ARID1A may be involved in the c-MYC pathway and modulates the ALL cell proliferation. Mutations of epigenetic regulators were also found in the polycomb complex (EZH2, EED, SUZ12), chromatin/nucleosome structure modifying proteins (CHD2, CHD3, CHD4), TET family proteins [TET1 (2 cases), TET2 (5 cases)] and histone modification proteins (HDAC1, SIRT1, BCOR, BRD8, lysine demethylase PHF2/KDM6A, histone acetyltransferase KAT6B).Transcription factors and p53/cell cycle pathway: a number of alterations of transcription factors essential for hematopoietic and lymphoid differentiation were noted including the lineage regulator PAX5 (5 missense, 3 indels) and ETV6 (6 cases, 3 were frameshift indel and 1 was a splice-site mutations). In addition, mutations were also found in other lineage transcription factors (IKZF2, IKZF3, EBF1), WT1 (6 cases, including 3 indels and 1 stop-gain mutations), RUNX family member [RUNX2 (7 cases), RUNX1 (1 case)], ERG1 (3 cases), GATA1/3 (1 case each) and CTCF. Somatic mutations of genes involved in the p53 pathway occurred in 18 patients, including TP53, ATM and the kinases that regulate p53 activities (HIPK1, HIPK2). Germline TP53 pathogenic variants were found in these 2 patients.Taken together, we extensively interrogated the mutational landscape of a large cohort of pediatric ALL samples by exome and targeted resequencing. This study provides a detailed mutational portrait of pediatric ALL and gives new insights into the molecular pathogenesis of this disease. DisclosuresKantarjian:Amgen: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Transcriptome Analysis Revealed the Entire Genetic Understanding of Pediatric Acute Myeloid Leukemia with a Normal Karyotype

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. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

NGS-Based Copy Number Analysis in 1,185 Patients with Myeloid Neoplasms

BackgroundCopy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms.Materials and MethodsWe enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%.ResultsTo obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions.Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy.ConclusionWe obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. [Display omitted] DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Clinical Significance of Mutations and Copy Number Lesions on Prognosis of Patients with MDS after Unrelated Bone Marrow Transplantation

Background:Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders of hematopoietic stem cells, characterized by defective hematopoiesis and dysplasia of multiple blood lineages. Patients with MDS could achieve complete remission only by allogeneic hematopoietic stem cell transplantation (HSCT). However, because of its high mortality and morbidity, long-term survival is accomplished only in the half of the patients, underscoring the importance of accurate prognostication before the therapeutic choice. For this purpose, several systems, such as the International Prognostic Scoring System, are being successfully applied to predicting patients' clinical outcomes. Advanced molecular diagnostics of recent years might further improve the prediction. Nevertheless, originally established based on the data from patients who are untreated or only supportively treated, existing systems may not always be applied properly to the prediction of outcomes of the patients who are treated by HSCT.Methods:We enrolled 790 patients with MDS who were treated by unrelated bone marrow transplantation between 2006 and 2013 through the Japan Marrow Donor Program. Oncogenic variants and copy number alterations were identified by targeted-capture sequencing of peripheral blood-derived DNA using RNA baits designed for 69 known or putative driver genes in myeloid neoplasms and 1,674 single nucleotide polymorphisms.Results:The median age at HSCT and observation period were 51 years old (16-66) and 1106 days (48-6018), respectively. At the time of transplant, 29%, 34%, 24%, and 5.2% of the cases were diagnosed as low-risk MDS, high-risk MDS, secondary acute myeloid leukemia, and myelodysplastic / myeloproliferative neoplasms, respectively, while the disease subtype was unknown for the remaining 8.2% of the cases. Mutations were observed in 73% of the patients, where U2AF1 was most frequently mutated (13.8%), followed by RUNX1 (12.9%), ASXL1 (12.8%), TP53 (12.4%), and NRAS (7.1%). The mean number of mutations was 2.0 per patient with a mean allelic burden of 43.8%. Sequencing data were successfully used for sensitive detection of CNVs and copy-neutral loss-of-heterozygosity (LOH) (or uniparental disomy; UPD). Among the most frequent lesions were 7q LOH, complex karyotype-like CNVs as defined by 3 or more CNVs excluding UPD in targeted sequencing), 5q LOH, trisomy 8, and 17p LOH, observed in 15.5%, 12.9%, 10.1%, 7.0%, and 6.8% of the patients, respectively. Mutations in TP53, CBL, and EZH2 significantly co-occurred with LOH in 17p, 11q, and 7q, with odds ratios of 190, 75.5, and 11.7, respectively. On the basis of these findings, we combined frequently identified LOH lesions with associated mutations for further analyses of survival. In univariate analysis of overall survival (OS), 9 lesions were significantly associated with shorter OS; TP53 mutation and/or 17p LOH (TP53 / 17p LOH) (HR 2.76, P = 1.6 x 10-14), CSNK1A1 / 5q LOH (HR 2.66, P = 2.13 x 10-12), CBL / 11q LOH (HR 2.42, P = 3.88 x 10-7), EZH2 / 7q LOH (HR 2.29, P = 1.46 x 10-12), NRAS mutations (HR 1.86, P = 2.0 x10-4), ETV6 / 12p LOH (HR 1.83, P = 2.84 x 10-5), 1q gain (HR 1.73, P = 0.0037), 20p LOH (HR 1.57, P = 0.030), and FLT3 mutations (HR 1.53, P = 0.027). The number of these unfavorable lesions significantly correlated with OS (P=8.9 x 10-16). Specifically, those with at least one mutation showed a significantly shorter OS, compared to those with none of these mutations (HR 2.54, P<2.0 x 10-16). TP53 / 17p LOH was the most unfavorable among the 9 lesions by multivariate analysis (HR 1.97, P=1.6 x 10-14). Multivariate analysis with clinical factors revealed that the presence of at least 1 of the 9 lesions was independently associated with poor OS (HR 2.05, P<2.0 x 10-16), together with well-known clinical factors negatively affecting OS, including red blood cell transfusion before HSCT (HR 1.93, P=0.0036), 3 or more grade of the performance status at HSCT (HR 1.92, P=1.6 x 10-4), and older age (HR 1.69, P=2.0 x 10-4). The presence of at least one lesion negatively affected OS irrespective of the presence of complex karyotype-like CNVs.Conclusions:The present study highlights the clinical significance of somatic mutations and CNVs in MDS cases treated by HSCT. Our findings suggest that the novel set of lesions identified in this study could be successfully used for the prediction of outcome in MDS in the setting of stem cell transplantation. DisclosuresKataoka:Yakult: Honoraria; Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria. Kanda:Otsuka Pharmaceutical: Honoraria, Research Funding. Makishima:The Yasuda Medical Foundation: Research Funding. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Distinctive Genetic Features of Plasma Cells in POEMS Syndrome

Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles.Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing.Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman’s variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5).WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases).To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case.We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM.Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Whole-Genome Sequencing of Primary Central Nervous System Lymphoma and Diffuse Large B-Cell Lymphoma

IntroductionPrimary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin's lymphoma. Although most cases (~95%) show histology of diffuse large B-cell lymphomas (DLBCLs), PCNSL shows very different biological and clinical characteristics from systemic DLBCL. Nevertheless, our knowledge about the molecular pathogenesis of PCNSL and genetic differences between both lymphomas are still incomplete.MethodTo obtain a comprehensive view of the genetic alterations, including mutations in non-coding regions as well as structural variants (SVs), we performed whole-genome sequencing (WGS) of 22 PCNSL cases. Subsequently, to unravel the genetic differences between PCNSL and systemic DLBCL, we re-analyzed WGS data from systemic DLBCL cases (N = 47) generated by the Cancer Genome Atlas Network (TCGA) and Cancer Genome Characterization Initiative (CGCI) using our in-house pipeline. The mean depth of WGS for tumor samples were 49X and 37X for PCNSL and DLBCL cases, respectively. Whole-exome sequencing (WES) was also performed for an additional 37 PCNSL cases to reliably capture driver alterations and also to analyze mutational signatures in PCNSL, which were compared to those obtained from the WES data for DLBCL from TCGA (N = 49).ResultsWGS identified 10.5 and 5.6 mutations per mega-base on average in PCNSL and DLBCL, respectively. We first explored the density of somatic mutations and identified 64 and 33 genomic loci showing significantly high mutation densities in PCNSL and DLBCL, respectively. In PCNSL, most of these loci corresponded to known targets of somatic hypermutations (SHMs) induced by activation-induced cytidine deaminase (AID), including those for IG genes (IGK, IGH and IGL), BCL6, and PIM1, as well as those for known driver genes, such as MYD88 and CD79B. Although most of the hypermutated regions were overlapped between PCNSL and DLBCL, some regions were differentially affected by hypermutations between both lymphoma types. For example, BCL2 and SGK1 loci were frequently affected by SHMs in germinal center B-cell (GCB) DLBCL, while not in PCNSL. In terms of non-coding driver mutations, we identified frequent mutations in a PAX5 enhancer region in 8/22 (36%) of PCNSL and 18/47 (38%) of DLBCL cases.SVs were common in both lymphoma types, where 104 (PCNSL) and 57 (DLBCL) SVs were detected per sample. SV clusters were identified in 34 (PCNSL) and 13 (DLBCL) regions, of which several clusters were commonly seen in both PCNSL and DLBCL, and included IG loci, BCL6, FHIT, TOX and CDKN2A. In PCNSL, SVs were clustered within the loci for known targets of SHMs, such as BCL6, BTG2 and PIM1. As was the case with somatic mutations, the SV cluster corresponding to BCL2 was only seen in DLBCL. We then analyzed these clustered breakpoints for their proximity to known sequence motifs targeted by AID (CpG and WGCW). Breakpoints of SVs found in the targets of SHMs, including PIM1, BCL6, BTG2 and BCL2, showed an enrichment at or near the CpG, supporting the involvement of AID in the generation of these SVs. By analyzing these SV clusters, we identified several novel driver genes in PCNSL. For example, WGS and WES identified an enrichment of breakpoints of deletions (7/22) and loss-of-function mutations (6/37) in GRB2, strongly indicating its tumor suppressor role in PCNSL.We also analyzed pentanucleotide signatures of mutations in coding sequences detected by WES of PCNSL and DLBCL, taking into consideration the two adjacent bases 3’ and 5’ of the substitutions as well as transcription strand biases. Two predominant mutational signatures were identified in PCNSL: the AID signature characterized by C>T mutations within the WRCY motif targeted by SHMs and the age-related signature involving C>T transition at CpG dinucleotides. For DLBCL, an additional signature (signature 17 according to Alexandrov et al.) was detected as well, which had been reported in DLBCL with an unknown mechanistic basis.ConclusionsComprehensive genomic analyses of a large cohort of PCNSL and DLBCL cases have revealed the major targets of somatic mutations and SVs, including novel driver genes. In both PCNSL and systemic DLBCL, an enhanced AID activity is thought to be associated with generation of both SHMs and SVs, although the activity and targets of AID seem to substantially differ between both lymphoma types, suggesting distinct pathogenesis therein. DisclosuresKataoka:Boehringer Ingelheim: Honoraria; Yakult: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Genetic Landscape and Clonal Evolution Following 5-Aza Therapy in Patients with High-Risk Myelodysplastic Syndromes

Genetic Landscape and Clonal Evolution Following 5-Aza Therapy in Patients with High-Risk Myelodysplastic Syndromes