ALBERT

Description: Background: Acquired aplastic anemia (AA), the prototypical bone marrow failure syndrome, is inferred to result from immune-mediated destruction of hematopoietic progenitors, as most patients respond to immunosuppressive therapies. Clonal hematopoiesis in AA is evident in the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells in as many as half of patients and by identification of uniparental disomies involving 6p (6pUPD) chromosome in 13% of cases. In addition, "clonal transformation", as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) is a serious long-term complication in 10-15% AA patients. Methods: We performed targeted deep sequencing and SNP array-based copy number (CN) analysis of peripheral blood- or granulocyte-derived DNA from 439 patients with AA (280 from US and 159 from Japanese cohorts) for a panel of 103 candidate genes, chosen because they are known to be frequently mutated in myeloid neoplasms. Germline DNA was available for 288 out of 439 patients and was used to confirm the somatic origin of mutations. Whole exome sequencing (WES) was performed in 52 cases. Where serial samples were available, the chronology of detected mutations was also investigated. Results: Targeted deep sequencing provided highly concordant results between the US and Japanese cohorts; approximately one third of AA patients had mutations in genes commonly affected in myeloid neoplasms, and about one third of patients in whom mutations were identified had multiple mutations. Multi-lineage involvement of mutations was confirmed in 6 cases using flow-sorted bone marrow samples. However, compared to myeloid neoplasms, mutations in AA were at much lower variant allele frequencies (VAFs) (

Description: Abstract 273 Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms showing a frequent transition to acute myeloid leukemia. Although they are discriminated from de novo AML by the presence of a preleukemic period and dysplastic cell morphology, the difference in molecular genetics between both neoplasms has not been fully elucidated because of the similar spectrum of gene mutations. In this regards, the recent discovery of frequent pathway mutations (45∼90%) involving the RNA splicing machinery in MDS and related myeloid neoplasm with their rare mutation rate in de novo AML provided a novel insight into the distinct molecular pathogenesis of both neoplasms. Thus far, eight components of the RNA splicing machinery have been identified as the targets of gene mutations, among which U2AF35, SF3B1, SRSF2 and ZRSR2 show the highest mutation rates in MDS and CMML. Meanwhile, the frequency of mutations shows a substantial variation among disease subtypes, although the genetic/biological basis for these differences has not been clarified; SF3B1 mutations explain 〉90% of the spliceosome gene mutations in RARS and RCMD-RS, while mutations of U2AF35 and ZRSR2 are rare in these categories (〈 5%) but common in CMML (16%) and MDS without increased ring sideroblasts (20%). On the other hand, SRSF2 mutations are most frequent in CMML (30%), compared with other subtypes (

Description: The phase 3 trial HOVON-50 was designed to evaluate the effect of thalidomide during induction treatment and as maintenance in patients with multiple myeloma who were transplant candidates. A total of 556 patients was randomly assigned to arm A: 3 cycles of vincristine, adriamycin, and dexamethasone, or to arm B: thalidomide 200 mg orally, days 1 to 28 plus adriamycin and dexamethasone. After induction therapy and stem cell mobilization, patients were to receive high-dose melphalan, 200 mg/m2, followed by maintenance with α-interferon (arm A) or thalidomide 50 mg daily (arm B). Thalidomide significantly improved overall response rate as well as quality of the response before and after high dose melphalan. Best overall response rate on protocol was 88% and 79% (P = .005), at least very good partial remission 66% and 54% (P = .005), and complete remission 31% and 23% (P = .04), respectively, in favor of the thalidomide arm. Thalidomide also significantly improved event-free survival from median 22 months to 34 months (P 〈 .001), and prolonged progression free from median 25 months to 34 months (P 〈 .001). Median survival was longer in the thalidomide arm, 73 versus 60 months; however, this difference was not significant (P = .77). Patients randomized to thalidomide had strongly reduced survival after relapse. This trial was registered on www.controlled-trials.com as ISRCTN06413384.

Description: Abstract 2697 Adult T cell leukemia (ATL) is an aggressive peripheral T-cell neoplasm highly resistant to conventional therapies. The development of ATL is thought to be initiated by immortalization of T cells by human T cell leukemia virus type I (HTLV-I) infection in early childhood, followed by accumulating genetic hits during a long latency period, which eventually cause neoplastic transformation of T-cells. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL, except for the role of tax protein in T-cell immortalization and alterations of other genes, including TP53, p16 and TCF8. So, in order to understand the genetic basis of ATL, we performed whole genome sequencing of paired-normal DNA from an ATL patient. In total 77 non-silent somatic mutations were detected, among which a TET2 mutation (R1261C) immediately drew our attention. TET2 mutations are found in a wide variety of myeloid malignancies in high frequency and implicated in their pathogenesis. The TET families of proteins are thought to be involved in the epigenetic regulation of gene expression through catalyzing conversion of 5'-methyl cytosine to 5'-hydroxymethyl cytosine, which are supposed to be further converted to unmethylated cytosine. One of the recent interests in TET2 mutations was the recent report of frequent TET2 mutations in peripheral T cell neoplasms, including angioimmunoblastic T-cell lymphomas, peripheral T-cell lymphomas not otherwise specified, as well as other B cell neoplasms, which is in agreement with the observation in TET2-deficient mice, which showed an expansion of bone marrow progenitor pools involving immature lymphoid populations, indicating that deregulated epigenetic machineries could be also involved in the development of mature lymphoid neoplasms. To explore this hypothesis, 144 samples of ATL were screened for mutations in TET2 and other epigenetic regulators commonly mutated in myeloid malignancies, including DNMT3A, IDH1/2 and spliceosome genes, using target deep sequencing. We found 16 TET2 mutations in 13 out of the 144 ATL cases (9%) together with other mutations, which was similar to the frequencies reported in other peripheral T cell neoplasms. Deep sequencing allowed for accurate estimation of allelic burden of these mutations. In most cases, TET2 mutations were found in the major tumor populations, indicating their early origin during clonal evolution. In addition, DNMT3A (2%), IDH1/2 (1%), SF3B1 (2%), ZRSR2 (1%) were also mutated, although at lower frequencies. In conclusion, we found TET2 mutation through whole genome sequencing. TET2 mutations were relatively common in ATL. Together with other mutations frequently found in myeloid malignancies, our finding provided an intriguing insight into the role of deregulated DNA methylation in the pathogenesis of ATL and also suggested that a common mechanism may underlie between the two very different blood cancers. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5084 Background: Waldeyer's ring (WR) is circular band of lymphoid tissue located at the opening of the respiratory and digestive tract. There is controversy as to whether WR should be considered as a nodal or extranodal site. In this study, we conducted retrospective analyses of WR involving MZLs (WR-MZLs) to identify their clinical features, treatment, prognosis- favor nodal or extranodal. And we want to get additional information about specific organ relationship between WR-MZL and other MALT sites. Patients and Methods: From 1987 to 2010, 124 patients who were histologically confirmed as a MZL arising from head and neck (H&N) area was including WR (47 patients) were reviewed. Primary orbit and ocular adnexa MZL patient was excluded this data collection. Results: The male/female ratio of the 47 patients was 23 to 24. The median age was 53 years (range, 17–77). The commonly involving sites were tonsil (53.2%) followed by nasopharyn (40.4%). Fourteen patients (29.7%) were accompanied with extra-WR area MALT site- gastrointestinal tract (14.9%), ocular and adnexa (12.8%). Ann Arbor stage I/II disease was present in 50% (23 out of 46). Thirty-nine patients were categorized into the low/low-intermediate risk group (84.8%) according to International Prognostic Index (IPI). Complete and partial remissions were achieved in 18 (78.3%) and 2 (8.7%) of the 23 stage I/II patients. In 23 patients with stage III/IV, CR and PR were achieved in 15 (65.2%) and 4 (17.4%), respectively. The median progression-free survival (PFS) was 3.6 years (95% CI, 2.4–4.8 years). The estimated 5-year overall survival (OS) was 88%. Compared with H&N MALT site MZLs, WR-MZLs were significantly poor in PFS and OS (P=0.005 and P=0.007). Conclusion: WR-MZLs were also an indolent disease like general MZL. But, WR-MZL patients presented with more advanced stage and poorer survivals than other site H&N MZL patients. Therefore, even though WR has been considered histologically extranodal MALT organ, clinically it is closer to nodal MZL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4623 Recently, clinical importance of disease status by computed tomography (CT) before treatment in chronic lymphocytic leukemia (CLL) is increasing. The present study was conducted to evaluate whether splenic volume (SV) by CT would predict the clinical outcome in CLL patients. SV were measured in 61 Rai II-IV CLL patients who underwent CT scan before chlorambucil therapy. SV was reconstructed and measured from the cross-sectional CT images by using 3 dimensional Voxar analytical volume software. Median SV was 721.3 cm3 (223.7-3576.6 cm3) is the most significant separated cut-off point on progression-free survival (PFS) and overall survival (OS). Low SV group was associated with longer PFS and OS than high SV group (p

Description: Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Nearly 40% of patients still relapse after present first-line therapies and once the relapse occurs, the long-term survival rates decrease, ranging from 21% to 34%. As for the pathogenesis of AML relapse, the recent development of massively parallel sequencing technologies has provided a new opportunity to investigate comprehensive genetic alterations that are involved in tumor recurrence of adult AML. However, little is known about the molecular details of relapsed pediatric AML. Methods In order to reveal the clonal origin and the major mutational events in relapsed pediatric AML, we performed whole exome sequencing of 4 trio samples from diagnostic, relapsed and complete remission phases using Illumina HiSeq 2000. Copy number abnormalities were also detected using whole exome sequencing. Subsequently, deep sequencing of identified mutations was performed to evaluate intra-tumor heterogeneity and the clonolocal history of relapsed clones. Results Whole-exome sequencing of 12 samples from 4 patients were analyzed with a mean coverage of more than x100, and 95 % of the targeted sequences were analyzed at more than x20 depth on average. A total of 98 somatic mutations were identified, where mean number of non-silent mutations was higher at relapsed phase than at the time of diagnosis (14.0/case vs 10.5/case) (p=0.270). Assessment of clonality using variant allele frequencies of individual mutations suggested that some mutations were subclonal mutations, consisting of intra-tumor heterogeneity both at the time of diagnosis and at relapse. In all 4 patients, relapsed AML evolved from one of the subclones at the initial phase, which was accompanied by many additional mutations including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process of leukemia recurrence. Forty-six out of the 98 mutations were specific either at the time of diagnosis (n = 16) or at relapse (n = 30). Relapse-specific mutations and copy number changes were found in several genes including known drivers such as NRAS and CREBBP. These mutations were further investigated in an extended cohort of relapsed pediatric AML samples using targeted sequencing to evaluate their prevalence. In some cases, AML relapse may accompany a dynamic clonal change. For example, some bona fide driver mutations, such as KRAS mutations, that were predominant at the time of diagnosis disappeared in relapsed samples. Discussion Whole exome sequencing unmasked clonal structure of primary and relapsed pediatric AML, which helped to understand the underlying mechanism of relapse in pediatric AML. Our results suggested that pediatric AML has intra-tumor heterogeneity and subclonal mutations such as NRAS and CREBBP occurring in one of the subclones could drive the AML relapse. Disclosures: No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in 〉 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.

Description: Immune cells show quick responses to infection. Many studies concerning cellular and humoral immunity have focused on the regulation of mature lymphocyte function. However, recent studies revealed that the early stage of hematopoiesis plays an important role in the immune system. In bone marrow, hematopoietic stem/ progenitors (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide, the ligand of Toll-like receptor 4, hematopoietic stem cells enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis almost arrests. Little has been known how early T lymphopoiesis in thymus changes. Previously, we cloned signal-transducing adaptor protein-2 (STAP-2) as a c-fms/M-CSFR interacting protein, and found STAP-2 in T lymphocytes or macrophages is crucial for immune responses. The function of STAP-2 is generally recognized under inflammatory condition, interacting with a variety of signaling or transcriptional molecules. We reported that STAP-2 binds to STAT5 for regulation of T cell proliferation, and in macrophages, STAP-2 combines with MyD88 and IκB kinase to activate NF-κB and enhances the production of IL-6 and TNFα. In this study, the effects of STAP-2 on early T progenitors were evaluated using gene-modified mice. All experimental procedures were conducted under specific pathogen-free conditions, according to protocols approved by Institutional Animal Care and Use Committees of Osaka University. We first evaluated the expression level of STAP-2 in murine thymus with quantitative PCR. STAP-2 mRNA was ubiquitously observed all through T cell development, including the CD4- CD8- double-negative (DN) stage. To test the influences on T lymphopoiesis, we generated knock-out and transgenic mice (Tg) that are modified STAP-2 gene expression. In Tg mice, STAP-2 was overexpressed under the control of the Lck proximal promoter. The promoter could drive expression of the inserted cDNA in T lineage cells from the late of DN 2 stage (CD44+ CD25+). We found that thymus was significantly enlarged in Tg mice (46.7 ± 11.15 mg in WT vs 88.1 ± 25.2 mg in Tg), while the number of T lymphocytes in periphery was comparable to wild-type mice (WT). Results from flow cytometric analysis showed STAP-2 enhanced the percentages of DN2 and DN3 (CD44- CD25+) T progenitors, and the actual numbers of DN2, DN3, DN4 (CD44- CD25-) and CD4+ CD8+ double-positive subpopulations. There were no differences between control and knock-out mice in thymus and peripheral bloods. When Lin- Sca1+ cKitHigh HSPC derived from Tg mice were cultured with Delta-like 1-transduced OP9 stromal cells (OP9-DL1) under T cell generation condition, the development of DN4 cells was accelerated (26.9 ± 6.2 % in WT vs 35.2 ± 4.1 % in Tg). Co-cultures from Lin- CD44+ CD25- cKitHigh early T cell progenitors showed the same tendency. These results indicate that STAP-2 regulates the proliferation and differentiation of T progenitors during DN3 to DN4 stage. To elucidate the signaling regulated by STAP-2, microarray experiment with DN3 T progenitors was conducted. The bioinformative approach with Ingenuity Pathway Analysis showed the canonical pathways related with IL-12 signaling, 4-1BB (CD137) signaling and helper T cell differentiation were significantly influenced. Interestingly, we found that STAP-2 affected the distribution of functional T lymphocytes. The ratio of helper CD4+ cells to suppressor CD8+cells in peripheral bloods was lower in Tg mice than that in WT. In summary, we found that STAP-2 regulates the early T lymphopoiesis in thymus. DN2 to DN4 stages of T progenitors increased in STAP-2 transgenic mice, and STAP-2 promoted the differentiation in vitro. Moreover, STAP-2 affected the cell decision in development to helper CD4+ cells or suppressor CD8+ cells. Our study indicates the up-regulation of STAP-2 under inflammatory condition might be crucial for immune response at the early stage of T lymphopoiesis. Further study would clarify the precise molecular mechanisms of the enhancement of T lymphopoiesis by STAP-2. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 458 MDS and related disorders comprise a group of myeloid neoplasms characterized by deregulated blood cell production and a predisposition to AML. Although currently, a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS, because many of them are also found in other myeloid malignancies and roughly 20% of MDS cases have no known genetic changes. So, in order to clarify a complete registry of gene mutations in MDS and identify those discriminate MDS from other myeloid neoplasms, we performed whole-exome sequencing of 29 cases showing myelodysplasia. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 41 occurred in recurrent gene targets, which not only included a spectrum of known gene targets in MDS, such as TET2, EZH2, NRAS/KRAS, RUNX1, TP53 and DNMT3A, but also affected previously unknown genes that are commonly mapped to the RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery. To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms (N=582) using a high-throughput mutation screen of pooled DNA followed by confirmation/identification of candidate mutations. In total, 219 mutations were identified in 209 out of the 582 specimens of myeloid neoplasms. Mutations of the splicing machinery were highly specific to diseases showing myelodysplastic features, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML (N=151) and MPD (N=53). The mutations among 4 genes, U2AF35 (N = 37), SRSF2 (N = 56), SF3B1 (N = 79) and ZRSR2 (N = 23), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 8), PRPF40B (N = 7), U2AF65 (N = 4) and SF1 (N = 5). Interestingly, mutations in the former three genes showed clear hot spots, indicating a gain-of-function nature of these mutations. On the other hand, two thirds of the ZRSR2 mutations are nonsense or frameshift changes causing premature truncation of the protein. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, and commonly affected those components of the splicing complex that are engaged in the 3' splice site recognition during RNA splicing, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. In fact, when transduced into HeLa cells, the recurrent S34F U2AF35 mutant induced the increase in the production of unspliced RNA species and elicited the activation of the nonsense mediated decay pathway. Functionally, the U2AF35 mutants seemed to cause deregulated stem cell functions, because CD34(−) KSL cells transduced with various U2AF35 mutants invariably showed reduced chimerism in competitive reconstitution assay. In accordance with this, the S34F U2AF35 mutant lead to suppression of cell growth in a variety of cell types, including HeLa cells, in which expression of the mutant induced a G2/M cell cycle arrest and increased apoptosis. In conclusion, whole-exome sequencing unexpectedly revealed the high frequency of the splicing pathway mutations in MDS and related myeloid neoplasms, providing the first evidence indicating that compromised RNA splicing by gene mutations are responsible for human pathogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.