ALBERT

Description: 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.

Description: Background: Therapy for patients (pts) with high risk and/or relapsed or refractory AML remains unsatisfactory. Retrospective studies have demonstrated activity of fludarabine, cytarabine, granulocyte colony stimulating factor and idarubicin (FLAG-IDA) as salvage therapy in pts with relapsed or refractory AML. Furthermore, a recent randomized trial has indicated high complete remission (CR) rates with improved relapsed-free survival when FLAG-IDA is administered as frontline induction chemotherapy (Burnett et al. J Clin Oncol 2013). Therefore, since January 2011, we have employed FLAG-IDA as first line therapy in pts with high risk AML (i.e. poor risk cytogenetics, antecedent myeloproliferative neoplasm or myelodysplastic syndrome, or therapy-related AML), or as first salvage in pts with primary refractory or relapsed AML, in an attempt to improve CR rates and permit more patients with AML to advance to allogeneic hematopoietic stem cell transplantation (alloSCT). Methods: A retrospective review was conducted of the 62 consecutive patients with high risk AML or primary refractory or relapsed AML treated with FLAG-IDA between January 2011 to December 2013 at the Princess Margaret Cancer Centre to determine the CR rate and overall survival (OS) associated with FLAG-IDA remission induction chemotherapy. Results: Baseline characteristics of the patients are listed in Table 1. Fourteen pts received FLAG-IDA as first induction, whereas 48 pts received FLAG-IDA as salvage (39 as first salvage and 9 as second salvage). The overall CR rate (i.e. CR + CR with incomplete platelet recovery [CRi]) using FLAG-IDA as frontline therapy was assessed in 13 patients, as one pt died during induction therapy and therefore, was not evaluable. Of the 13 evaluable patients, all achieved CR or CRi. The overall CR rate for the salvage induction group was 73% (i.e. 31% CR and 42% CRi). The CR duration was censored at time of transplant. The CR duration for pts receiving FLAG-IDA as first induction was 3 mos (range, 0-15 mos). For pts receiving FLAG-IDA as salvage therapy, the CR1 duration for primary refractory AML pts was 6 mos (range, 2-58 mos) and CR2 duration for relapsed AML pts was 4 mos (range, 1-12 mos). 76% of patients (n=10) who received frontline FLAG-IDA induction chemotherapy, and achieved CR/CRi, had a donor identified, but only 40% of those pts underwent alloSCT. 85% of pts (n=30) who received salvage FLAG-IDA, and achieved CR/CRi, had a donor identified, but only 53% of those pts proceeded to alloSCT. The length of hospital stay during the first FLAG-IDA induction was 33 days (range, 17-96 days), whereas the length of hospital stay for salvage FLAG-IDA induction was 43 days (range, 10-305 days). Fourteen percent of pts in the first induction group were admitted to the ICU during their induction, compared to 17% of pts in the salvage induction group. The median ICU stay was 39.5 days and 14 days, respectively. There was a 14% death rate during FLAG-IDA induction for both groups. The median follow up time from diagnosis for both groups was 15.28 mos (range, 2-70.4 mos). Overall survival at 1 and 2 years in the upfront FLAG-IDA induction group was 65% and 41%, respectively, while OS at 1 and 2 years for the salvage FLAG-IDA group was 60% and 35%, respectively. Conclusions: The toxicities associated with FLAG-IDA induction, including induction death rates and ICU admission rates, are acceptable and similar in the untreated and heavily pre-treated groups. FLAG-IDA induction can result in durable CR rates, permitting patients with high risk AML or patients with primary refractory or relapsed AML to proceed to allogeneic transplantation. Table 1: Patient Characteristics Front-LineN=14 SalvageN=48 Median age, y (range) ≥70y (%) ≥60y (%) 65.5 (21-76) 2 (14%) 10 (71%) 50 (18-76) 2 (4%) 10 (21%) Gender 7M : 7F 22M : 26F Secondary/Therapy-related Prior MDS Prior MPN 14 (100%) 2 (14%) 2 (14%) 17 (35%) 8 (17%) 2 (4%) Cytogenetic risk group Good Intermediate Poor 0 4 (28%) 10 (71%) 3 (6%) 28 (58%) 17 (35%) Molecular abnormalities cKit mutated FLT3-ITD mutated 0 1 (7%) 2 (4%) 5 (10%) Median no. prior treatment regimens (range) 0 1 (1-2) Prior chemotherapy regimen Daunorubicin + cytarabine NOVE-HiDAc Other NA NA NA 43 (90%) 11 (23%) 3 (6%) Disease status Primary refractory Relapsed CR1 duration

Description: Chemokines mediate the signaling and migration of T cells, but little is known about the transcriptional events involved therein. Microarray analysis of CXC chemokine ligand (CXCL) 12−treated T cells revealed that Wnt ligands are significantly up-regulated during CXCL12 treatment. Real-time polymerase chain reaction and Western blot analysis confirmed that the expression of noncanonical Wnt pathway members (eg, Wnt5A) was specifically up-regulated during CXCL12 stimulation, whereas β-catenin and canonical Wnt family members were selectively down-regulated. Wnt5A augmented signaling through the CXCL12-CXCR4 axis via the activation of protein kinase C. Moreover, Wnt5A expression was required for CXCL12–mediated T-cell migration, and rWnt5A sensitized human T cells to CXCL12-induced migration. Furthermore, Wnt5A expression was also required for the sustained expression of CXCR4. These results were further supported in vivo using EL4 thymoma metastasis as a model of T-cell migration. Together, these data demonstrate that Wnt5A is a critical mediator of CXCL12-CXCR4 signaling and migration in human and murine T cells.

Description: Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

Description: 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

Description: Key PointsThe high-resolution structure of the complex disulfide-bonded TIL′E′ (D′) region of VWF is presented. The major factor VIII binding site is localized around a flexible region on the TIL′ domain.

Description: 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.

Description: 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

Description: Background: Acute myeloid leukemia (AML) with t(8;21) or inv(16) is commonly referred to as core-binding factor AML (CBF-AML). Although this group represents a favorable cytogenetic AML subgroup, 30-40% of these patients nevertheless relapse after standard intensive chemotherapy. The incorporation of high-dose cytarabine for postremission therapy has substantially improved the outcome of CBF-AML patients, especially when administered as 2-4 repetitive cycles. Here we present retrospective data from a single centre, on this favourable AML subgroup. Methods: We analyzed retrospectively the outcome of 80 sequential patients with CBF-AML (46 t(8;21), 34 inv(16)/t(16;16)) treated over a 13 year period from 2000-2012. The median age was 48 years (range 20-80) with a median white cell count of 13x109/L(range 1-426x109/L). All patients underwent induction chemotherapy consisting of daunorubicin (60 mg/m2/d x 3 days) and continuous infusion cytarabine (100 or 200 mg/m2/d x 7 days, for ages

Description: Background: Although classified by WHO 2008 as belonging to the category “Acute myeloid leukemia and related precursor neoplasms”, Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) presents as an acute leukemia (AL) only in a minority of cases. There are only few studies describing the comprehensive immunophenotypic pattern of BPDCN in the bone marrow. Furthermore, given the rarity of this hematologic malignancy optimal frontline therapy is unclear. Patients and Methods: This retrospective analysis evaluates the diagnostic flow cytometry pattern and outcome of 9 patients who were diagnosed with BPDCN at the Princess Margaret Cancer Centre between December 2008 and June 2014. A four tube 10-color flow cytometry (FCM) panel has been used to correctly make the diagnosis of BPDCN in 6 patients, whereas a 5-colour panel was used in the remaining patients in conjunction with immunohistochemistry. The following markers were included in the10-color panel: Tube 1: CD65 FITC, CD13 PE, CD14 ECD, CD33 PC5.5, CD34 PC7, CD117 APC, CD7 A700, CD11b A750, CD16 PB, and CD45 KO; Tube 2: CD36 FITC, CD64 PE, CD56 ECD, CD33 PC5.5, CD34 PC7, CD123 APC, CD19 A700, CD38 A750, HLA-DR PB, and CD45 KO; Tube 3: CD71 FITC, CD11c PE, CD4 ECD, CD33 PC5.5, CD34 PC7, CD2 APC, CD10 A700, CD235a A750, CD15 PB, and CD45 KO; Tube 4:nuclear (n) TdT FITC, cytoplasmic (cyt.) MPO PE, CD14 ECD, CD33 PC5.5, CD34 PC7, cyt.CD79a APC, cyt.CD22 A700, CD19 A750, cyt.CD3 PB, and CD45 KO. Results: Median age was 66 years (range, 25 to 91 years); 3 patients were over the age of 70 years. Fifty-six percent were males. All presented with skin lesions and 78% presented each with lymphadenopathy and bone marrow involvement. Cytogenetics were poor-risk in 2 patients, intermediate-risk in 3 and unknown or inconclusive in 4. By 10-color FCM, leukemic cells were in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA-DR, CD71, but negative for CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD34, CD64, CD65, CD235a. Other markers, such as cyt.MPO, cyt.CD3, cyt.CD22 and nTdT were negative, while dim cyt.CD79a was seen in 3 cases. CD7 expression was found in 5 cases, whereas CD2 and CD117 were found in single cases only. BM involvement by BPDCN leukemic cells ranged from 27% to 92% of the marrow cellularity. Skin involvement showed dense infiltrate of cells with blastoid morphology and characteristic grenz zone. Seven patients received front-line induction therapy with HyperCVAD with an overall response rate of 86% (4 complete remissions (CR), 2 unconfirmed CRs). One patient died of multi-organ failure during induction. Three of 6 responders underwent planned allogeneic hematopoietic cell transplantation (HCT); 1 patient has since died of acute graft versus host disease (GVHD), whereas 2 are alive in remission with chronic GVHD, 12 and 14 months post transplant with complete donor chimerism. One transplant ineligible patient relapsed 22 months after achievement of CR1. Median follow-up of all patients was 12 months with a overall survival at 1 year of 59.3% for the entire group. Patients who underwent allogeneic HCT had overall survival at 1 year of 66.7% and for the chemotherapy group was 27.8% at 1 year.(p=0.34). Conclusion: An accurate diagnosis of BPDCN can be made by 10-colour FCM using a 4-tube acute leukemia panel. BPDCN demonstrates a characteristic pattern of antigen expression . Although front-line induction chemotherapy with HyperCVAD can yield high CR rates, allogeneic HCT should be performed in first CR for transplant eligible patients, as this appears to be required for long term durable remissions. For transplant ineligible or relapsed BPDCN patients, there is an unmet need for novel therapeutic agents. Disclosures Porwit: Beckman-Coulter: Speakers Bureau. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.