ALBERT

Beschreibung: Abstract 404 To characterize the genomic events associated with distinct subtypes of AML, we used whole genome sequencing to compare 24 tumor/normal sample pairs from patients with normal karyotype (NK) M1-AML (12 cases) and t(15;17)-positive M3-AML (12 cases). All single nucleotide variants (SNVs), small insertions and deletions (indels), and cryptic structural variants (SVs) identified by whole genome sequencing (average coverage 28x) were validated using sample-specific custom Nimblegen capture arrays, followed by Illumina sequencing; an average coverage of 972 reads per somatic variant yielded 10,597 validated somatic variants (average 421/genome). Of these somatic mutations, 308 occurred in 286 unique genes; on average, 9.4 somatic mutations per genome had translational consequences. Several important themes emerged: 1) AML genomes contain a diverse range of recurrent mutations. We assessed the 286 mutated genes for recurrency in an additional 34 NK M1-AML cases and 9 M3-AML cases. We identified 51 recurrently mutated genes, including 37 that had not previously been described in AML; on average, each genome had 3 recurrently mutated genes (M1 = 3.2; M3 = 2.8, p = 0.32). 2) Many recurring mutations cluster in mutually exclusive pathways, suggesting pathophysiologic importance. The most commonly mutated genes were: FLT3 (36%), NPM1 (25%), DNMT3A (21%), IDH1 (18%), IDH2 (10%), TET2 (10%), ASXL1 (6%), NRAS (6%), TTN (6%), and WT1 (6%). In total, 3 genes (excluding PML-RARA) were mutated exclusively in M3 cases. 22 genes were found only in M1 cases (suggestive of alternative initiating mutations which occurred in methylation, signal transduction, and cohesin complex genes). 25 genes were mutated in both M1 and M3 genomes (suggestive of common progression mutations relevant for both subtypes). A single mutation in a cell growth/signaling gene occurred in 38 of 67 cases (FLT3, NRAS, RUNX1, KIT, CACNA1E, CADM2, CSMD1); these mutations were mutually exclusive of one another, and many of them occurred in genomes with PML-RARA, suggesting that they are progression mutations. We also identified a new leukemic pathway: mutations were observed in all four genes that encode members of the cohesin complex (STAG2, SMC1A, SMC3, RAD21), which is involved in mitotic checkpoints and chromatid separation. The cohesin mutations were mutually exclusive of each other, and collectively occur in 10% of non-M3 AML patients. 3) AML genomes also contain hundreds of benign “passenger” mutations. On average 412 somatic mutations per genome were translationally silent or occurred outside of annotated genes. Both M1 and M3 cases had similar total numbers of mutations per genome, similar mutation types (which favored C〉T/G〉A transitions), and a similar random distribution of variants throughout the genome (which was affected neither by coding regions nor expression levels). This is consistent with our recent observations of random “passenger” mutations in hematopoietic stem cell (HSC) clones derived from normal patients (Ley et al manuscript in preparation), and suggests that most AML-associated mutations are not pathologic, but pre-existed in the HSC at the time of initial transformation. In both studies, the total number of SNVs per genome correlated positively with the age of the patient (R2 = 0.48, p = 0.001), providing a possible explanation for the increasing incidence of AML in elderly patients. 4) NK M1 and M3 AML samples are mono- or oligo-clonal. By comparing the frequency of all somatic mutations within each sample, we could identify clusters of mutations with similar frequencies (leukemic clones) and determined that the average number of clones per genome was 1.8 (M1 = 1.5; M3 = 2.2; p = 0.04). 5) t(15;17) is resolved by a non-homologous end-joining repair pathway, since nucleotide resolution of all 12 t(15;17) breakpoints revealed inconsistent micro-homologies (0 – 7 bp). Summary: These data provide a genome-wide overview of NK and t(15;17) AML and provide important new insights into AML pathogenesis. AML genomes typically contain hundreds of random, non-genic mutations, but only a handful of recurring mutated genes that are likely to be pathogenic because they cluster in mutually exclusive pathways; specific combinations of recurring mutations, as well as rare and private mutations, shape the leukemia phenotype in an individual patient, and help to explain the clinical heterogeneity of this disease. Disclosures: Westervelt: Novartis: Speakers Bureau.

Beschreibung: Abstract 2105 The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. We hypothesized that common variants in genes involved in iron metabolism may modulate susceptibility or resistance to the development of iron deficiency in humans. To examine the association between single nucleotide polymorphisms (SNPs) in key genes involved in iron metabolism pathways, we previously performed a genome-wide association study using DNA collected from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤12 μg/L (cases) and controls (SF 〉100 μg/L in men, SF 〉50 μg/L in women). We now report on a multiethnic follow-up association study of HEIRS participants. Candidate SNPs were identified from our GWAS and the scientific literature. Population samples of whites, African Americans, Hispanics, and Asians from the U.S. and Canada were analyzed separately for association between SNPs and case-control status and each of seven quantitative outcomes including serum iron, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC), transferrin saturation, SF, serum transferrin receptor, and body iron. There were 1084 white (357 cases, 727 controls), 153 Asian (51 cases, 102 controls), 221 African American (77 cases, 144 controls) and 233 of 239 Hispanic individuals (79 cases, 160 controls) that passed quality control. For the African-American and Hispanic samples, ancestry proportions were estimated based on genotypes of ancestry informative markers. Regression analysis was used to examine the association between case-control status and quantitative serum iron measures and 1134, 1115, 1113 and 1134 SNP genotypes in the white, African-American, Hispanic, and Asian population samples, respectively. Model predictors included age, sex, the estimated ancestry proportion (for African American and Hispanic only), genotype, and measured covariates that showed nominally significant associations with the outcome. Three chromosomal regions showed evidence of association across multiple populations, including SNPs in the TF gene on chromosome 3q22, the TMPRSS6 gene on chromosome 22q12, and loci on chromosome 18q21. SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p=4.7×10−7) and was replicated in African Americans (p=0.0012).Twenty SNPs in the TF gene region were significantly associated with TIBC in the white sample (p

Beschreibung: Although the effects of type II-IFN (IFN-γ) on GVHD and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-α/β) remain unclear. We investigated this using type I-IFN receptor-deficient mice and exogenous IFN-α administration in established models of GVHD and GVL. Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed toward either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4+ T-cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this finding, administration of IFN-α during conditioning inhibited donor CD4+ proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor, respectively. This finding reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell-mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BM transplantation could be studied prospectively in patients at high risk of relapse.

Beschreibung: Abstract 236 Altered expression of microRNAs (miRNAs, a class of small regulatory RNAs) is associated with various types of cancers, including acute myeloid leukemia (AML). We showed previously that increased expression of miR-181a with or without miR-181b was associated with a favorable prognosis for patients with cytogenetically normal AML (CN-AML). However, the prognostic value of miR-181 expression in cytogenetically abnormal AML (CA-AML) remains elusive, even though CA-AML represents the majority of human AML. To investigate the association of expression signatures of miR-181 family members and of their potential target genes with outcome in patients with primary CA-AML, we employed two independent sets of 86 CA-AML patients to investigate the association of expression signatures of miR-181 family members with outcome. We also used four independent sets of 454 CA-AML patients to identify and validate a prognostic signature of miR-181 targets. In addition, we investigated the biological functions of miR-181a/b and target(s) in leukemia cell lines and in a leukemia mouse model. As with CN-AML, we found that both miR-181a and miR-181b expression signatures are significantly (P

Beschreibung: Abstract 672 Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and repairing DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells but previous attempts at gene therapy failed largely due to inadequate numbers of hematopoietic stem cells available for gene correction and autologous engraftment. Induced pluripotent stem cells (iPSC) constitute an alternate source of autologous cells, which are amenable to ex vivo expansion and genetic correction. While fibroblasts from a limited number of FA patients have been reported to fail to undergo reprogramming (Raya et al., Nature, 2009), reproducible observations and mechanistic studies ascertained in an extended panel of patient cells and murine knock-out models are lacking to date. We undertook direct reprogramming of ten unique human FA primary fibroblast samples of the FA-A, FA-C, FA-G, and FA-D2 complementation groups. Using standard four-factor reprogramming, no human FA iPSC colonies were obtained in cells defective in the FA pathway. By contrast, reprogramming of gene-corrected patient samples, augmented by hypoxia (5%O2), yielded multiple pluripotent iPSC lines, confirming a critical cell-intrinsic role of the FA pathway in reprogramming. To determine if gene-corrected FA iPSC could be therapeutically useful, we performed karyotype analyses and evaluated in vitro hematopoietic differentiation in three FA-A iPSC lines. These FA patient iPSC lines were karyotypically normal and showed a robust multilineage hematopoietic differentiation potential, resulting in erythroid and myeloid hematopoietic colony forming units to a similar degree as compared to normal donor iPSC controls. We hypothesized that the reprogramming resistance of FA cells is due to defective DNA repair and genomic instability. To explore the mechanisms of the reprogramming defect, we transduced wild type (wt) tail-tip fibroblasts (TTF) with the reprogramming vectors. We observed significantly increased FANCD2 foci formation during reprogramming (median percentage of FANCD2 foci: mock-transduced TTF 2.5%, reprogrammed TTF 20.5%, n=8, p

Beschreibung: Abstract 1400 BACKGROUND: Activating mutations of the FLT3 gene are common in AML, with approximately 25–30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis which can be modulated by the combination of co-operating mutations. In animal models, expression of FLT3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 12: 367). This is supported by in vivo models which demonstrate that the combination of the FLT3-ITD mutation with other genetic lesions leads to an acute leukemia in mice. AIMS: To identify novel genes co-operating with FLT3-ITD to induce acute leukemia by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with a FLT3-ITD background. METHODS: An autosomal dominant screen for FLT3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU mutagenised male mice homozygous for the FLT3-ITD knock-in allele to females homozygous for the FLT3-ITD knock-in allele. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state. Peripheral blood differential analysis was performed at 8, 12 and 16 weeks of age as well as immunophenotyping for the myeloid markers Mac1 and Gr1 and the progenitor marker c-kit. Analysis of a non-mutagenised cohort of mice indicated a sex-specific differential effect of FLT3-ITD on multiple blood cell parameters, so male and female data were analysed separately. Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers (Figure 1) and bred to FLT3-ITD homozygous knock-in mice to test for heritability of the phenotype (Figure 2). RESULTS: To date 150 G1 mice have been screened, leading to the identification of four heritable phenotypes (Designated pedigrees 12, 21, 24 and 37, Figure 1). All pedigrees were characterised by increased WBC counts compared to ‘unaffected’ mice (FLT3-ITD homozygous background) and by further expansion of the myeloid compartment. Preliminary analysis of the bone marrow from affected mice identified an increase in colony replating ability in response to GM-CSF, suggestive of a block in differentiation. Three out of the four pedigrees showed disease penetrance only in males, with several mice from pedigree 24 succumbing to disease (Figure 2). Whole exome sequencing is being undertaken to identify the sequence variants that segregate with the phenotype in each of the pedigrees. CONCLUSIONS: It is possible through mutagenesis to identify co-operating mutations with FLT3-ITD. Further characterisation of these pedigrees, the associated phenotype and identification of the associated mutation in each case, will provide important information regarding pathways that co-operate with FLT3-ITD in leukemogenesis. Translation of these findings to human AML will have potential implications for predicting the course of disease in AML patients and may indicate pathways that will be targets for new and complementary treatments in AML. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4+ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4+ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25+ Treg cells was similar to that achieved by in vivo depletion of all CD4+ T cells. Depletion of CD25+ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4+ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8+ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Beschreibung: Abstract 1462 Despite progress made in understanding the biology and risk-adapted treatment of AML, prognosis of older pts [age ≥60 years (y)] remains poor. We have shown that high expression of the BAALC gene bestows worse outcome in older CN-AML pts. Yet, the mechanism(s) by which BAALC affects response to therapy remain unknown. MicroRNAs (miRs) are small noncoding RNAs that hybridize to target mRNAs and inhibit their translation or promote degradation, resulting in downregulation of the corresponding proteins. Recently, miR-3151 was discovered embedded in intron 1 of BAALC. We thus hypothesized that miR-3151 might be co-expressed with BAALC and contributes alone or in concert with its host to poor prognostic impact in older CN-AML. MiR-3151 and BAALC expression were measured by quantitative real time (RT) PCR in pretreatment blood of 179 older CN-AML pts enrolled on Cancer and Leukemia Group B (CALGB) cytarabine/daunorubicin-based protocols. The expression values of miR-3151 and BAALC were normalized to the internal controls SNORD44 and RN18S1, respectively. MiR-3151 expression correlated only weakly with the expression of BAALC (Spearman correlation r= 0.3). For clinical and prognostic analyses pts were dichotomized into high and low miR-3151 and BAALC expressers at the median miR and gene transcript expression values and analyzed for other molecular prognosticators (FLT3 -ITD, FLT3 -TKD, CEBPA, IDH1, IDH2, NPM1, TET2, WT1 mutations and ERG expression). Gene (GEP) and microRNA (MEP) expression profiles associated with miR-3151 expression were derived, using Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, respectively. At diagnosis, higher miR-3151 expression was associated with lower % blood blasts (P =.02), FAB types M4 and M5 (P =.05), and wild-type NPM1 (P

Beschreibung: Relapsed or refractory (rel/ref) classical Hodgkin lymphoma (cHL) remains a clinical challenge, with limited effective treatment options available after stem cell transplantation. In a multicenter phase 2 study, the efficacy of lenalidomide in rel/ref cHL patients was evaluated at a dose of 25 mg/d on days 1-21 of a 28-day cycle. Patients remained on lenalidomide until disease progression or an unacceptable adverse event (AE) occurred. Thirty-eight cHL patients were enrolled with a median of 4 (range, 2-9) prior therapies; 87% had undergone prior stem cell transplantation and 55% of patients did not respond to their last prior therapy. Of 36 evaluable patients, responses were 1 complete remission (CR), 6 partial remissions (PRs), and 5 patients with stable disease (SD) for ≥ 6 months resulting in an International Working Committee (IWC) objective overall response rate (ORR) of 19% and a cytostatic ORR of 33%. Decreased chemokine (CCL17 and CCL22) plasma levels at 2 weeks were associated with a subsequent response. The treatment was well tolerated, and the most common grade 3/4 AEs were neutropenia (47%), anemia (29%), and thrombocytopenia (18%). Four patients discontinued lenalidomide because of rash, elevated transaminases/bilirubin, and cytopenias. We provide preliminary evidence of lenalidomide's activity in patients with rel/ref cHL, and therefore exploration of lenalidomide in combination with other active agents is warranted. This trial is registered at www.ClinicalTrials.gov as NCT00540007.

Beschreibung: Abstract 227 The histone methyl-transferase EZH2 is frequently mutated in germinal-center derived diffuse-large B cell lymphoma (DLBCL) and follicular lymphoma. We and others have demonstrated that these mutations in tyrosine 641 of EZH2 represent a dominant gain-of-function, enhancing the capability to trimethylate H3K27 in combination with wildtype activity (Yap et al, Blood 2011; Sneeringer et al, PNAS 2010). To further test if these EZH2 mutations can be driver mutations in lymphomagenesis we have now generated and characterized a mouse line transgenic for EZH2 Y641F (the most commonly observed mutant form). Transgenic mice were created on the C57/Bl6 background by pronuclear microinjection of a construct where EZH2 Y641F is expressed from a lymphoid specific Emu-lck promoter. Spleen cells isolated from the transgenic mice displayed a global increase in H3K27me3 when analyzed by Western Blot. However none of the mice showed signs of lymphoma even when observed for prolonged periods of time. As EZH2 mutations often coincide with other mutations in lymphoma, we also combined the expression of EZH2 Y641F with the over-expression of Myc by crossing one of the transgenic lines with Emu-myc transgenic mice. In this combination model, we observed a dramatic acceleration of lymphoma development with the combination of Myc and EZH2 Y641F as compared to Myc alone (Myc alone (n=26) 137.5 days, Myc+EZH2 Y641F (n=20) 51 days, p 〈 0.0001 Log-Rank (Mantel-Cox) Test). The resulting lymphomas are characterized by a generalized massive lymphadenopathy, splenomegaly (spleen weight 0.416 +/− 0.133 g), bone marrow infiltration (72 +/− 8 %) and lymphocytosis (WBC 138.5 +/− 77.1 ×1000/μl). They show histological features of high-grade disease, and initiate disease in secondary recipients. In contrast to lymphomas observed with Emu-myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2 Y641F, presented with a mature B cell phenotype (B220+IgM+). This shift in differentiation can also be observed in the bone marrow even before development of overt lymphomas. While Myc transgenics mainly show an increase in the B220+IgM−compartment, this is shifted towards B220+IgM+ in double transgenics. Cell cycle analysis by in vivo Brdu incorporation before disease onset shows a drastic increase in the fraction of cells in cycle in the splenic B cells of double transgenic mice (43.3 and 44.9 % cells in S phase) as compared to mice transgenic for Myc alone (10.6 % and 14.2 % cells in S phase). Southern Blot analysis using a probe specific for JH4 showed more than one clonal rearrangement in a majority of the lymphomas in the double-transgenic mice. In summary, EZH2 Y641F can collaborate with Emu-myc in high efficiency lymphoma induction. This new murine lymphoma model now provides a powerful tool to study global changes in the epigenome caused by this frequent mutation and may be a useful system for testing novel treatments. Disclosures: No relevant conflicts of interest to declare.