ALBERT

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: 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: 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.

Description: BACKGROUND: Activating mutations of the FLT3 gene are common in acute myeloid leukemia (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, the severity of which, can be modulated by the combination of co-occuring 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. 2007, 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 the development of an acute leukemia in mice (Chen et al, Genes Dev. 2013, 27: 1974). AIMS: To identify and characterise novel genes that alter Flt3-ITD induced MPN by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with an Flt3-ITD homozygous knock-in 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 homozygous Flt3-ITD females. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state (compared to that induced by Flt3-ITD alone). Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers and bred to Flt3-ITD homozygous knock-in mice to test for heritability of the phenotype. Where pedigrees were generated demonstrating heritable phenotypes, multiple affected and unaffected littermates were subject to exome sequencing and analysis to identify a list of candidate gene mutations segregating with the disease phenotype. RESULTS: 150 G1 mice were screened, leading to the identification of four pedigrees with heritable phenotypes marked by an exacerbated MPN. Exome sequencing has identified a short list of 3 genes for one pedigree (pedigree 37) that includes a mutation in Neurofibromatosis 1 (Nf1), a gene known to be involved in the induction of juvenile myelomonocytic leukemia and frequently lost in AML (Parkin et al,Clin Cancer Res 2010, 16:4135). In another pedigree (pedigree 24) we identified a mutation in Ndufa10 as the single candidate segregating with the phenotype (Figure 1A-B). Ndufa10 encodes a subunit of the mitochondrial respiratory complex I, which is the first and largest complex in the mitochondrial electron transport chain. Importantly, germline mutations in this gene lead to a complex I deficiency syndrome in humans, indicating that it is a critical subunit of this complex. We hypothesise that mutation of Ndufa10 leads to altered cellular metabolism in hematopoietic stem and progenitor cells which contributes to exacerbation of the MPN, possibly through an alteration in production of reactive oxygen species and a shift in the balance between glycolysis and oxidative phosphorylation. Breeding of the Ndufa10 mutation onto a non Flt3-ITD background shows that action of the mutation is not dependent on the presence of Flt3-ITD, as these mice also have altered blood counts, including increased WBC (Figure 1C). CONCLUSIONS: It is possible to identify mutations that exacerbate Flt3-ITD induced MPN through mutagenesis and an efficient blood screening strategy. In addition, using this strategy, we have identified novel mutations that act independently of Flt3-ITD to induce changes in the haematological compartment. Translation of these findings to human AML may indicate pathways that will be targets for new and complementary treatments in AML. Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4048 Poster Board III-983 Introduction Iron deficiency is the most common nutritional disorder in the world with an estimated two billion affected persons. Although commonly considered environmental in origin, the existence of multiple genetic disorders of iron metabolism in man, rodents and other vertebrates suggest a genetic contribution to iron deficiency. Methods: The Hemochromatosis and Iron Overload Screening (HEIRS) Study is a multi-center, multi-ethnic study in which transferrin saturation (TS), serum ferritin (SF), and HFE mutations were determined in 101,168 adults. To identify genomic locations associated with iron deficiency, we performed a genome-wide association study (GWAS) using DNA collected from white HEIRS Study participants who had SF ≤ 12 μg/L (cases) and an equal number of white controls (SF 〉 100 μg/L in men, SF 〉 50 μg/L in women) frequency-matched to cases by sex and geographic location. Men aged ≥ 25 y and women ≥ 50 y were included in both groups. Tissue body iron, an index of iron deficiency, was estimated from serum transferrin receptor (sTfR) and SF. Genotyping was performed with the Illumina HumanCNV370K Beadchip platform. Quality control filters excluded single nucleotide polymorphisms (SNPs) or samples with 〉 5% missing genotypes, SNPs showing heterozygosity or Hardy-Weinberg deviations (P 100 μg/L and women with SF 〉 50 μg/L). Results The GWAS genomic control parameter was not significantly different from 1.0. There were 392 cases (96 men) and 390 controls (96 men) in the HEIRS subset GWAS with average age (SD) of 59 (10) y and 61 (11) y, respectively. Geometric mean SF (minimum, maximum), and mean (SD) for sTfR and tissue body iron in the HEIRS subset were 7.5 (1.2, 12) μg/L, 6.4 (3.77) mg/kg and -2.0 (2.50) for cases and 141 (51, 881) μg/L, 3.0 (0.98) mg/kg and 10.8 (2.5) for controls. After quality control tests, GWAS analysis included genotype data for 331,060 SNPs in 734 individuals (364 cases, 370 controls). For the VA replication population there were 67 male and 11 female cases, and 136 male and 27 female controls for whom DNA was successfully prepared; the average age (SD) was 68 (12) y for cases and 65 (11) y for controls. Regression analysis identified seven SNPs within four independent regions that replicated associations found in the GWAS (GWAS P

Description: Abstract 1599 Poster Board I-625 The greatest obstacle to routine clinical testing of gene expression levels has been the lack of reproducibility of currently used methodologies, such as quantitative reverse transcriptase PCR (qRT-PCR) and microarray expression profiling. While these assays are useful for retrospective analyses of batched samples, they cannot be used for upfront evaluation of individual patients (pts) for molecular risk and treatment assignment. To overcome this barrier, we tested a recently developed, high throughput, PCR-independent, digital quantification technology, the nCounter system (Nanostring® Technologies). This system counts individual mRNA molecules, rather than measuring non-linear fluorescence generated by PCR-amplified targets (eg qRT-PCR). Using 72 AML samples and spike-in controls, we and collaborators demonstrated that the nCounter system is highly reproducible, sensitive, and accurate to femtomolar concentrations (Payton, J, et al. JCI 119:1714-26; Geiss, G, et al. Nat Biotech 26:317-25). Here we validated this technology using an independent set of 101 pts with a diagnosis of de novo cytogenetically normal AML. At diagnosis, pts presented with FAB subtypes M0, M1, M2, M4, M5(A, B), had a median age of 43 years (range 19-59), median white blood count of 28.5× 103/μL (range 1.4-273.0), median of 69% BM blasts (range 22-95) and median of 65% PB blasts (range 0-97). Paired BM and PB specimens were available for 27 pts; blast percentages were ≥ 20% for all paired specimens. We used the nCounter system to measure mRNA abundance (‘counts‘) of 27 genes whose expression correlates with clinical and/or pathological criteria, including 3 genes associated with prognosis (BAALC, ERG, MN1), and control/housekeeping genes (GAPDH, ABL, Actin). Briefly, mononuclear cells from pretreatment BM or PB were enriched on Ficoll-Hypaque gradients and RNA was isolated using Trizol reagent; 100ng of total RNA was assayed in triplicate by nCounter according to the manufacturer's protocols. The nCounter results demonstrated substantial reproducibility, with a median CV [coefficient of variation, (standard deviation/mean *100)]