Citations

Abstract 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<4.4×10−5); six SNPs were replicated in other ethnicities (p< 0.01). SNP rs10904850 in the CUBN gene on 10p13 was significantly associated with serum iron in the African-American sample (P=1.0×10−5). Mutations in the TMPRSS6 gene have been implicated in iron-refractory iron deficiency anemia through linkage studies. We found a novel SNP in TMPRSS6 that was associated with serum iron in whites and replicated in African Americans, suggesting a role for this SNP in increasing the risk of iron deficiency in affected persons. Our results confirm known associations with iron measures and give evidence of their role in different ethnic groups, a unique aspect of this study, suggesting origins in a common founder. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4105 Background: JAK2 mutations are found in approximately 99% of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). It is currently assumed that other mutations relevant to JAK signaling contribute to the pathogenesis of JAK2 mutation-negative myeloproliferative neoplasms (MPN). The same might hold true for some cases of “idiopathic erythrocytosis” associated with subnormal serum erythropoietin level (sEpo). LNK is a plasma membrane-bound adaptor protein whose function includes inhibition of wild-type and mutant JAK2 phosphorylation. LNK exon 2 mutations were recently described in two patients with JAK2V617F-negative ET or PMF. Both mutations involved the LNK pleckstrin homology (PH) domain; one was a 5 base-pair deletion and missense mutation leading to a premature stop codon (603_607delGCGCT; 613C>G) and the other a missense mutation (622G>C; glutamic acid to glutamine substitution; E208Q). Objectives: i) To estimate the prevalence of LNK mutations in chronic and blast phase MPN ii) To determine if LNK mutations are mutually exclusive of other MPN-associated mutations iii) To test the hypothesis that LNK mutations might contribute to the pathogenesis of JAK2 mutation-negative PV or otherwise unexplained erythrocytosis Methods: LNK mutation analysis was performed on bone marrow or blood cells using modified primers for amplifying the SH2 and PH domains, using previously published methods (Oh et al. Blood First Edition Paper, prepublished online April 19, 2010; DOI 10.1182/blood-2010-02-270108). Results: LNK mutation studies were performed in 172 patients; paired chronic-blast phase samples were analyzed in 26 cases. Diagnoses in the 172 study patients were as follows: 78 chronic-phase MPN (39 JAK2V617F-positive PV, 25 PMF and 14 ET) enriched for TET2, IDH, JAK2V617F, or MPL mutations, 61 blast-phase MPN (41 blast-phase PMF, 11 blast-phase PV and 9 blast-phase ET), 25 JAK2V617F-negative PV and 8 “unexplained erythrocytosis” associated with subnormal sEpo and negative for Epo receptor (EPOR) mutations. Ten novel heterozygous LNK mutations, all but one affecting exon 2 in the PH domain, were identified: 6 missense mutations involving codons 215, 220, 223, 229 and 234, one nonsense mutation involving codon 208 (622G>T leading to glutamic acid to a stop codon substitution; E208X), one synonymous mutation involving codon 208, and 2 deletion mutations involving exon 2 (685-691_delGGCCCCG) or exon 5 (955_delA). LNK mutations were most frequent in blast-phase MPN, occurring in 6 (9.8%) of the 61 informative patients; chronic-phase sample analysis in 4 of these revealed the same mutation in only one case. Mutant LNK was detected in chronic-phase samples only in 2 additional patients with blast-phase MPN. Among the 8 LNK-mutated blast-phase MPN cases, 7 had blast-phase PMF. The clinical phenotype in the remaining 2 LNK-mutated patients was that of an isolated erythrocytosis without overt features of PV. JAK2V617F was documented in 3 and IDHR140Q in 1 LNK-mutated patients. Conclusions: LNK mutations i) target an exon 2 ‘hot spot' in the PH domain spanning residues E208-D234, ii) might be most prevalent in blast-phase PMF iii) are not mutually exclusive of JAK2 or IDH mutations and iv) might be part of the “missing link” in the pathogenesis of JAK2 mutation-negative “idiopathic” erythrocytosis. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: We previously reported that Lenalidomide combined with R-CHOP (R2CHOP) has significant activity in non-GCB (non-germinal center B-cell) subtype of DLBCL as defined by immunohistochemistry (IHC) using Hans algorithm (J Clin Oncol. 2015 Jan 20;33(3):251-7). However, defining non-GCB subtype of DLBCL by IHC as opposed to the "gold standard" gene expression profiling (GEP) is subject to technical limitations and its prognostic value has not reproduced in some recently published studies. In addition, recent reports questioned prognostic value of GEP in prospective clinical trials as opposed to initial reports from retrospective case series. To address these concerns, we assessed outcomes by cell of origin (COO) as determined via GEP using NanoString Lymphoma Subtyping Test (LST) on formalin-fixed paraffin-embedded (FFPE) tissue on patients enrolled in a phase 2 study of R2CHOP and an independent comparison cohort of patients treated with RCHOP alone. Methods: Eligible patients for R2CHOP phase 2 study MC078E were adults with newly diagnosed, untreated, stages II-IV CD20 positive DLBCL. Patients received oral lenalidomide 25 mg days 1-10 with standard dose R-CHOP every 21 days for 6 cycles. All patients received pegfilgrastim on day 2 of each cycle and aspirin prophylaxis. DLBCL molecular subtype was determined by GEP using NanoString LST, a multiplexed digital gene expression assay performed on the nCounter® Dx Analysis System and reported as GCB vs ABC vs unclassified. A comparison cohort of RCHOP treated DLBCL patients was generated from the prospectively enrolled Mayo Clinic component of the Lymphoma SPORE Molecular Epidemiology Resource (MER). MER patients meeting the same inclusion criteria as MC078E with available tissue were selected based via matching on age, stage, and IPI to the MC078E patients. Event-free survival was defined as time from diagnosis (MER) or study registration (MC078E) to progression, re-treatment, or death. EFS24 was defined as being event-free at 24 months. Associations between COO and outcome were assessed via log-rank tests due to censoring for EFS24. Results: 50 MC078E R2CHOP patients enrolled between 2008 and 2013,124 matched MER RCHOP patients diagnosed between 2003 and 2012 were assessed for COO. Median follow up in 70 patients still alive was 7.9 years (1.9-13.0) in RCHOP treated patients and 4.4 years (1.9-5.5) for 24 in R2CHOP treated patients still alive. Median age was 65 years (range 35-89) in RCHOP treated patients and 69 years (22-87) in R2CHOP patients (p=0.485). 53 RCHOP treated patients (43%) and 31 R2CHOP treated patients (62%) had intermediate-high or high IPI (p=0.021). COO was GCB, ABC, and unclassified DLBCL subtype in 80, 31 and 13 patients in the 124 RCHOP cohort and 33, 13 and 4 patients in the R2CHOP treated patients, respectively (see table). EFS24 was inferior for ABC (48%) compared to GCB DLBCL 71% in MER RCHOP treated patients (p=0.013). In contrast, in R2CHOP treated pts, EFS24 was not different between ABC vs GCB DLBCL 69% vs 67% (p= 0.674). Conclusion: Cell of origin classification of DLBCL determined by NanoString LST remains prognostic in a series of prospectively enrolled DLBCL patients treated with RCHOP. R2CHOP shows promising efficacy in ABC DLBCL as defined by NanoString LST, where the addition of lenalidomide to RCHOP appears to mitigate the negative impact of an ABC molecular subtype on the outcome. Table Table. Disclosures Nowakowski: Morphosys: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Research Funding. Maurer:Kite Pharma: Research Funding; Celgene: Research Funding. Storhoff:NanoString Technologies, Inc.: Employment, Other: Stock option. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock option. Gandhi:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction The phagocytic activity of macrophages is regulated by activating ("eat") and inhibitory ("do not eat") signals. Under normal physiologic conditions, the ubiquitously expressed cell surface antigen CD47 suppresses phagocytosis by binding to signal regulatory protein alpha (SIRPα) on macrophages. It is hypothesized that overexpression of CD47 by cancer cells enables immune evasion. Blockade of CD47 results in phagocytosis of cells bearing "eat" signals and primes effective anti-tumor T cell responses. TTI-621(SIRPαFc)is a soluble recombinant fusion proteinconsisting of the CD47 binding domain of human SIRPα linked to the Fc region of human IgG1designed to both: 1) block the CD47 "do not eat" signal, and 2) engagemacrophage Fcγ receptors with IgG1 Fc to enhance phagocytosis and antitumor activity.In vitro, TTI-621 binds to normal human cells, platelets, a wide range of human primary tumor cells and cell lines, but only minimally to human erythrocytes. TTI-621 selectively promotes macrophage-mediated phagocytosis of hematologic and solid tumors over that observed with normal monocytes, and exhibits antitumor activity in xenograft mouse models. Methods A first-in-human, phase 1, open label, multicenter study (NCT02663518) is ongoing to evaluate the safety and tolerability, and to identify the maximum tolerated dose of TTI-621 in patients (pts) with relapsed/refractory lymphomas using a 3+3 dose-escalation design. Once the optimal dose has been determined in the dose-escalation phase, multiple expansion cohorts will be enrolled comprising pts with various relapsed/refractory hematologic malignancies. Assessments include peripheral receptor occupancy, serum cytokine levels, pharmacokinetics, and immunogenicity. Eligible pts are adults with advanced, measurable, hematologic malignancies, who have progressed on standard anticancer therapy or for whom no other approved therapy exists. Pts are required to have baseline hemoglobin ≥10 g/dL, platelets ≥75 x 109/L, and be transfusion- and growth factor-independent. Pts with cutaneous T-cell lymphoma, high-grade lymphoma, and acute promyelocytic leukemia are excluded. TTI-621 is administered IV once weekly at protocol-defined doses. Treatment may continue until disease progression or unacceptable toxicity. Results Eleven pts (6M/5F, age 21-72 years) have been enrolled as of the data cut-off date of 28 July 2016. Lymphoma diagnoses included Hodgkin (N=4), diffuse large B cell (DLBCL) (N=4), follicular (N=2), and mantle cell (N=1). Treatment has been reasonably well tolerated by pts in the 0.05 mg/kg (N=3), 0.1 mg/kg (N=3), and 0.3 mg/kg (N=5) dose cohorts. The majority of pts experienced mild to moderate infusion-related events. Hemoglobin levels have remained stable or improved with treatment. Transient, dose-dependent decreases in platelets and leukocytes occurred in the hours following infusion in all pts without clinical sequelae. The 0.3 mg/kg dose was associated with reversible, dose-limiting toxicity (DLT) in 2 of 5 pts: one pt with G3 elevated ALT/AST and G4 platelet count, and a second pt with G4 platelet count who was transfused. Dosing at 0.2 mg/kg is now being explored. Aside from the DLTs and 2 non-DLT G3 platelet count (all in 0.3 mg/kg cohort), treatment-related adverse events have been ≤G2. CD47 receptor occupancy increased with each cohort, peaking at the end of infusion and remaining detectable 24 hrs after the 1st infusion in Cohort 3. Macrophage-associated cytokines, including MIP-1α and MIP-1β, increased during the 4 hrs after infusion. Six pts continue to receive weekly infusions of TTI-621; one pt with DLBCL and another with FL have experienced progression-free intervals of 161 and 70 days, respectively. Conclusions TTI-621 has been reasonably well tolerated. Pts retained stable hemoglobin levels consistent with minimal drug binding to erythrocytes. Manageable, dose-dependent thrombocytopenia was likely due to increased phagocytic clearance of platelets. TTI-621 binds to CD47+ cells in a dose-dependent manner, potently yielding increases in cytokines associated with augmented phagocytic activity. Enrollment continues at the 0.2 mg/kg dose level; updated data will be provided at the meeting. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Chen:Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Consultancy, Research Funding, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Merck: Consultancy, Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. O'Connor:Bristol Myers Squibb: Research Funding; Spectrum: Research Funding; TG Therapeutics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; TG Therapeutics: Research Funding. Johnson:Trillium Therapeutics: Employment. Irwin:Hoffmann La Roche: Employment, Equity Ownership; Trillium Therapeutics: Employment, Equity Ownership. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Sievers:Seattle Genetics: Employment, Equity Ownership; Trillium Therapeutics: Employment, Equity Ownership; MEI Pharma: Consultancy.

Abstract Background Cytotoxic agents are a well-established risk factor for therapy related myeloid neoplasms (t-MN). The incidence of t-MN in patients (pts) with autoimmune diseases (AID) treated with cytotoxic and immuno-modulatory agents are not well described in the literature. Most case series assess an MDS/AML population and co-occurrence with an AID. To our knowledge, there exists no large case-control studies evaluating a primary AID population that developed therapy-related MDS or AML, and the relationship to detailed therapeutic intervention for AID. Our study queries the association of cytotoxic and immune-modulating agents to treat AID as risk factor for developing MDS or AML. Methods Between 2004-2014, 23,671 pts with an ICD-9 coded AID were seen at Mayo Clinic Arizona; 148 pts from this population had a concomitant coded diagnosis of MDS/AML. In an IRB approved, retrospective chart review we confirmed 61 cases meeting strict inclusion criteria. We performed a case-control match from the same primary AID population for age, gender, and autoimmune specific diagnosis at a 2:1 ratio. AML/MDS cases were compared to controls by use of chi-square test for frequency data. Odds ratios with 95% confidence interval were constructed using conditional logistic regression. Case incidence was compared to SEER data for the general population Results Of 61 cases 33 were MDS, 20 de-novo AML, and 8 MDS transformed to AML. Psoriasis 16 (26.2%) and rheumatoid arthritis 14 (23.0%) were the most common autoimmune diagnoses, followed by systemic lupus 11 (18%), Crohn's disease 7 (11.5%), and ulcerative colitis 5 (8.2%). 39 out of 61 cases (63.9%) received either a cytotoxic or immune modulating agent for AID, compared to 64/122 (52.5%) in the control group; p=0.140. 7/61 cases (11.5%) received both a cytotoxic and immune modulating agent as compared to 16/122 (13.1%) in controls; p=0.753. Overall, the case population received more agents than controls. No cytotoxic or immune-modulating treatment was documented in 16% of cases and 36% controls. Exposures to cytotoxic and immune-modulating agents were similar in both case and control cohorts. Exposure time was not statistically significant. Azathioprine was the only statistically significant agent exposure observed more often in the case cohort than control. Per SEER data, over a 10-year period in a general age-matched population that is comparable to our AID population cohort, we would expect 54 MDS and 35 AML cases. Table 1. Exposures and Timeline Cases (n=61) Controls (n=122) P value * Number of Systemic Agents, Total None 1 2 3 4+ - 10 (16.4%) 26 (42.6%) 12 (19.7%) 7 (11.5%) 6 (9.8%) - 44 (36.1%) 32 (26.2%) 31 (25.4%) 9 (7.4%) 6 (4.9%) 0.020 Exposure to Cytotoxic agent < 1 year 2-5 years 6-10 years 11 years or more Unknown (n=26) 7 (26.9%) 6 (23.1%) 5 (19.2%) 3 (11.5%) 5 (19.2%) (n=43) 5 (11.6%) 8 (18.6%) 6 (13.9%) 12 (27.9%) 12 (27.9%) 0.276 Exposure to Immune modulating agent < 1 year 2-5 years 6-10 years 11 years or more Unknown (n=20) 6 (30%) 6 (30%) 3 (15%) 1 (5%) 4 (20%) (n=37) 6 (16.2%) 10 (27.0%) 5 (13.5%) 3 (8.1%) 13 (35.1%) 0.661 Table 2. Treatments and Odds Ratio Odds Ratio 95% CI for OR P value * Cytotoxic OVERALLAzathioprine Cyclophosphamide Methotrexate Mitoxantrone 6-MP Mycophenolate 1.39 4.16 1.28 0.74 2.73 1.00 1.21 0.72, 2.67 1.29, 13.44 0.24, 6.89 0.29, 1.88 0.23, 33.0 0.23, 4.35 0.28, 5.21 0.322 0.017 0.773 0.528 0.429 0.999 0.803 Immune modulating agent OVERALLAnti-TNF Leflunomide Plaquenil 1.19 0.73 2.00 2.73 0.52, 2.73 0.24, 2.24 0.40, 9.91 0.80, 8.33 0.672 0.585 0.396 0.077 Discussion Treatment of AID does not seem to increase the incidence of MDS or AML. Furthermore, the incidence of myeloid neoplasm occurring in association with treatment of AID is not higher than expected in the general SEER population. However, the case cohort was exposed to more agents overall and more likely to have exposure to azathioprine than controls. Limitations of our study are associated with its retrospective nature, possible associated underrepresentation of t-MN, and autoimmune population profile. Nevertheless, this is the largest case study with detailed drug exposures with timelines for pts with AID, contributing to a better understanding regarding possible risk of developing MDS/AML in pts treated for AID. Confirmation of the same study at Mayo Clinic Rochester and Florida are ongoing. Disclosures Al-Kali: Novartis: Research Funding.

Abstract Abstract 2919 Background: Sideroblastic anemias can be either hereditary due to congenital mutations in factors critical for iron processing or heme biosynthesis, or acquired; acquired sideroblastic anemias may be induced by alcohol or medications, but are usually idiopathic. Pyridoxine, a form of vitamin B6, plays a critical role in heme synthesis as a cofactor for δ-aminolevulinic acid synthetase (ALAS). Some subtypes of congenital sideroblastic anemia, such as those associated with mutations in the ALAS2 gene encoding the erythrocyte-expressed isoform of ALAS, may respond to pyridoxine therapy at doses ranging from 5–500 mg/day. Anecdotal reports of improvement with pyridoxine therapy in cases of acquired idiopathic sideroblastic anemia (AISA) have led to widespread clinical use of this agent in patients with refractory anemia with ring(ed) sideroblasts (RARS) and refractory cytopenia with multilineage dysplasia associated with ring sideroblasts (RCMD-RS). However, there are no systematic studies of the effectiveness of pyridoxine in AISA. Methods: We reviewed clinical and laboratory data from 231 adult patients with marrow aspirate-proven AISA (i.e., RARS or RCMD-RS, based on 2001 WHO criteria) evaluated at our institution between 1994 and 2007. Responses to pyridoxine were assessed using 2006 International Working Group (IWG) standardized criteria for MDS (erythroid response with hemoglobin increase by '1.5 mg/dl). The relationship between response to pyridoxine and disease subtype or International Prognostic Scoring System (IPSS) stratification was assessed using χ2 test, using a p-value limit of <0.05 for statistical significance. Results: 86 of the 231 patients (42%) were treated with pyridoxine for an average of 19 months (range 1–114 months) at a mean dose of 167 mg/day (range 50–600 mg/day). Sufficient follow-up data to allow response evaluation were available from 74 (86%) of the 86 patients who received pyridoxine. Only 5/86 patients (6.8%) receiving pyridoxine met IWG response criteria for hematological improvement, but 3 of these 5 patients also received erythropoetin and 1 also received prednisone concomitantly with pyridoxine therapy. Therefore, only 1/86 (1.4%) patient's improvement in hemoglobin could be attributed to pyridoxine monotherapy. ALAS2 genotype data were not available from these 5 patients. The dose of pyridoxine was not associated with response to therapy (187.5 mg daily in responders vs. 157 mg daily in non-responders (p=0.60). Patients with RCMD-RS were more likely to be treated with vitamin B6 compared to patients with RARS (p=<0.001), possibly because of more severe anemia, but response to pyridoxine did not differ significantly between subtypes (3/49 vs. 2/25, response in RARS vs. RCMD-RS; p=0.76). Among the 74 evaluable patients, 3/46 patients in the low IPSS risk group responded to pyridoxine, compared to 2/24 of patients in the Intermediate-1 risk group and 0/4 in the Intermediate-2 risk group (p=0.82). Adverse effects associated with pyridoxine included new onset of irreversible symptomatic peripheral neuropathy in 2/86 patients (2.3%). Conclusions: Pyridoxine is commonly prescribed to patients with AISA in clinical practice, and this agent is often continued for a long period of time despite lack of evidence of objective response. Pyridoxine is an ineffective therapy in AISA that induces symptomatic peripheral neuropathy in some patients. Therefore, pyridoxine therapy should be limited to patients with known or suspected congenital mutations that confer pyridoxine responsiveness, and therapeutic trials should be brief to avoid adverse effects. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Allogeneic Hematopoietic cell transplantation (HCT) is potentially curative therapy for MDS and AML. Although the median age at presentation is >65 years, most patients receiving HCT for these disorders are young (<60years). With the availability of Reduced Intensity Conditioning (RIC) many older patients are able to undergo HCT. The older age group is the most rapidly increasing cohort in CIBMTR registry. We analyzed the outcomes of AML and MDS patients' ≥ 60 years who received allogeneic sibling or unrelated donor HCT at our institution between 2009 and 2014. We also evaluated the influence of patient-, disease-, and transplant related factors on outcomes. Methods: Prospectively collected data available in the BMT databases at Mayo Clinic Florida, Arizona and Rochester as well as individual patient charts were reviewed after IRB approval. Patients with MDS or AML in complete remission at the time of transplant were included; those with active disease, induction failure, or prior transplant were excluded. HCT-specific comorbidity index (HCT-CI) and the disease risk index (DRI- Armand et al. Blood 2012) was noted. Survival estimates were calculated using the Kaplan Meier method. Associations with survival were explored using single variable Cox-regression modeling and log-rank tests. Results: 80 patients were included; 68 AML and 12 MDS. 31 (39%) were male. Median age 66 years (range 60-75). 33%, 52%, and 13% patients had ECOG status of 0, 1, >2, respectively. Thirty-seven patients (46%) had low or intermediate DRI and 43(54%) had high DRI. Twenty-two (28%), 24 (30%), and 34 (43%) patients had HCT-CI of 0, 1, or >2. There were equal number of sibling and unrelated donors; 85% were full HLA match and 15% with 1 or 2 mismatches; mean age of the donors was 52years (range18-74). Transplant conditioning was fludarabine-containing reduced intensity regimens in 78 (97%), 2 patients received myeloablative conditioning. The source of stem cells was peripheral blood in all patients. A median of 5.87 x10^6 CD34/kg donor cells were infused (range 1.58-10.92 X10^6). All patients received calcineurin inhibitor based GVHD prophylaxis (tacrolimus 56%, cyclosporine 44%); 6 patients also received ATG. The median follow up of survivors was 13 months. Kaplan Meier estimate of overall survival at 100 days, 1 year, and 2 years was 90%, 64%, and 55%. Cumulative incidence of relapse at 6 months, 1 year, and 2 years was 19.5%, 22.3%, and 27.1%. Relapse free survival at 2 years was 50%. The non-relapse/ treatment related mortality at 6 months, 1 year and 2 years was 11.7%, 15.9% and 15.9%. The rate of acute grade 2-4 GVHD was 30% and chronic GVHD was 39%. Univariate analysis identified secondary disease (compared to de novo) and high DRI(compared to low/intermediate) to be associated with worse mortality - HR 2.54 (1.31-4.93; P = 0.006), and 2.31 (1.13-4.73; P = 0.021), respectively. Probability of overall survival at 6 months, 1 year, and 2 years for patients with de novo disease (n=57) vs. secondary disease (n=23) was 87.1% vs. 64.5%, 71.1 vs. 45.6%, and 63.2 vs. 32.6%, respectively; P= 0.006. There was no effect of donor age, donor type or degree of mis-match on the survival. Conclusion: Allogeneic HCT is feasible in older patients with MDS and AML utilizing reduced intensity conditioning regimens with acceptable outcomes that are comparable to published results in younger patients. Patients with high DRI and secondary disease have poorer overall survival as compared to those with de novo disease and low/intermediate disease risk index. These data support other studies that age alone does not negatively influence transplant outcomes. Disclosures No relevant conflicts of interest to declare.

Abstract Philadelphia-negative Myeloproliferative Neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis Primary (PMF) and secondary to PV and ET (PPV-, PET-MF); included are also some less characterized entities defined as unclassified MPN (U-MPN). Risk of arterial and venous thrombosis is increased in MPN patients, and thrombosis is one of most important causes of mortality and morbidity. The risk of venous thrombosis in unusual sites, such as splanchnic vessels (SVT), is particularly associated with MPN; SVT can lead to complications such as portal hypertension, esophageal and gastric varices, ascites and hepatic failure. A recent meta-analysis reported that a MPN is the underlying cause of portal vein thrombosis in 31.5% and of Budd Chiari syndrome in 40.9% of cases (Smalberg, 2012). A significant association of SVT with JAK2V617F mutated MPN was reported (Dentali, 2009) but study of other correlations has been hampered by heterogeneity of available patient cohorts comprising relatively small number of cases. We conducted a retrospective multicenter study collecting clinical and biological data of patients (pts) with SVT associated with MPN diagnosed according to WHO2008 criteria, aiming to describe patients’ characteristics, trends and prognostic factors, and their potential implications for clinical practice. Data were collected from 15 international hematology centers in the framework of IWG-MRT. We collected 475 cases of pts with portal, splenic or mesenteric vein thrombosis (75.2%) or Budd Chiari syndrome (24.8%) associated with MPN. In 32% of cases, simultaneous involvement of portal (69.1% of total thrombosis), splenic (30.5%) and mesenteric (25.3%) veins occurred, and in 1.7% they were associated with Budd Chiari syndrome. Frequency of MPN subtype: 38.1% ET (n=181), 34.9% PV (n=166), 16.2% MF (n=77), 10.8% U-MPN (n=51). Median follow-up 87.9 mo (range 0.5-430); female 61.3% (n=292; P<0.0001 vs male); median age at MPN diagnosis (dg) 44.4 y (range 12-90), at SVT dg 44.9 y (range 17-85). In 229 cases (48%) MPN and SVT dg were coincident, while in 104 (22%) SVT occurred before MPN dg (median 40 mo, range 5-335) and in 129 (27%) during MPN follow up (median 79 mo, range 5-394). JAK2V617F mutational status is available for 361 pts: 99% PV, 84.7% ET, 88.1% PMF and 92.9% U-MPN pts were JAK2V617F positive, with a mean allele burden of 56±27.4%, 33.1±25.5%, 39.3±19.4% and 23.8±11.9%, respectively. Erythropoietin-independent colonies (EEC) were present at diagnosis in 80/110 evaluated cases (72.7%), 38/47 PV (84.4%), 32/45 ET (71.1%), 8/11 PMF (72.7%) and 2/7 U-MPN (28.6%). A concurrent thrombophilic state was found in 38.9% of cases. A 12.3% of pts experienced a recurrence of SVT after a median of 29 mo (range 1-378.3) and 35.8% developed thrombosis in other sites (17.7% arterial, 19.3% venous). Esophageal varices were found in 70.6% from which 31.9% bled. MF transformation occurred in 32/166 PV (19%) and in 23/181 ET (13%) pts, with median time to progression of 122.3 mo (range 5.4-377.3) and 125.1 mo (range 39.3-255.3), respectively. Evolution to acute leukemia (AL) occurred in 12 pts (2.7%), of which 2 PMF, 6 PV and 4 ET. In 3 PV and 1 ET pts a PPV and PET-MF transformation occurred before AL. After SVT, 77% of pts received anticoagulation, 23.5% antiaggregant therapy and 1.5% both; 68.8% received cytotoxic drugs, 11.4% of pts were treated with trans jugular porto-systemic shunt. No differences in survival were noted with these approaches. Beta blocker therapy was used in 48.5% of pts and correlated with improved survival (p=0.041) At last follow up 70/473 pts (14.8%) died; causes of death are evolution to AL (16.4%), other cancers (14.5%), disease progression without AL (12.7%), SVT (10.9%), hepatic failure and venous thrombosis other than SVT (9.1% each), heart failure and arterial thrombosis (7.3% each), hemorrhage (5.5%), renal failure and infection (3.6% each). After 10 y follow up 8/166 PV (5%), 14/181 ET (8%), 14/77 PMF (18%) and 1/51 U-MPN (1.96%) pts died (p<0.01). Survival was significantly affected by occurrence of thrombosis other than SVT (p<0.0001) but not recurrence in splanchnic vessels (p=0.068). This large study confirms the strong association between JAK2V617F-mutated MPN and SVT and identifies the category of U-MPN as the prognostically more favorable; thrombosis at sites outside the splanchnic vasculature remains the leading cause of death. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4527 Introduction: The prognosis for pts with intermediate or high risk AML remains dismal, with relapse rates typically in the 60–80% range after treatment with chemotherapy alone. While allo-SCT can decrease the risk of relapse to 10–20%, widespread use of this modality is limited by relatively high rates of non-relapse mortality (NRM), often due to severe acute and/or chronic GVHD. Attenuation of severe GVHD, without a concomitant increase in relapse or NRM, should improve outcomes and result in cure of a larger fraction of AML pts. We therefore investigated the use of in-vivo T-cell depletion with r-ATG in pts with intermediate- or high-risk AML but without active leukemia at the time of allo-SCT. Patients and Methods: Pts (n = 43) were included in this retrospective analysis if they were between 18 and 65 years of age and had no evidence of active AML at the time of allo-SCT (see Table). All pts had 1 or more high-risk features: 1) adverse or intermediate risk cytogenetics (without NPM1 mutation if cytogenetically normal); 2) therapy-related or secondary AML; 3) high WBC count at diagnosis; 4) failure to achieve CR after 1 cycle of induction; or 5) not in CR1 at allo-SCT. Among the 43 pts, 10 received grafts from related donors, 14 from 10/10 matched unrelated donors (URDs), and 19 from mismatched URDs (9/10, n = 11; 8/10, n = 8). All pts received r-ATG according to institutional standard operating policy, with doses ranging from 2.5 – 10 mg/kg depending on donor type and degree of mismatch. All transplants were performed using PBSC. Additional GVHD prophylaxis included tacrolimus plus either methotrexate or mycophenolate mofetil. Results: The median age was 47 (range 20 – 65), and median follow-up for surviving pts is 12 (range 1 – 66) months. As of 8/5/11, 39 pts were alive, and 4 had died from multiorgan failure (n = 1), relapse (n = 1), GVHD (n = 1), and veno-occlusive disease (n = 1). The 2-year estimate of PFS is 84.7% (Fig. 1). The 2-year cumulative incidence of relapse is 6.8% (2 pts, days 97 and 147), and of non-relapse mortality 9.4%. Three pts developed severe (grades III-IV) acute GVHD by day 100 (cumulative incidence 4.6% at day 100) with no additional cases of severe acute GVHD beyond day 100. To date, 4 pts have developed moderate/severe chronic GVHD (cumulative incidence 16.8% at 2 yrs), with one death at day 344 related to complications of acute and chronic GVHD. CMV reactivation occurred in 29 pts (56%), with no deaths related to CMV. Three pts have reactivated EBV, with one case of PTLD (all treated with Rituximab). Conclusions: In this retrospective analysis of single center data, the inclusion of r-ATG in the GVHD prophylactic strategy appeared to significantly attenuate the incidence and severity of both acute and chronic GVHD. Although follow-up is relatively early, the incidence of relapse and NRM does not appear to be increased compared to contemporaneous pts treated without ATG. Given that almost half of the pts received grafts from mismatched URDs, this abrogation in risk of GVHD is significant and clinically relevant. While randomized studies are needed, these data suggest that in-vivo T-cell depletion with r-ATG ameliorates severe GVHD, without increasing relapse or non-relapse mortality, in AML pts without overt leukemia at the time of allo-SCT. Using this strategy, cure rates of 70 – 80% may be realistic and attainable for younger (</= age 65) AML patients who achieve a leukemia-free state and who have a reasonably matched related or unrelated donor. Disclosures: Reeder: Celgene: Research Funding; Millennium Pharmaceuticals Inc.: Research Funding; Novartis: Research Funding.