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Introduction: The optimal treatment approach for systemic AL amyloidosis remains unclear. Autologous stem cell transplant (ASCT) is the only modality associated with long term survival, but failure to show survival benefit in randomized clinical trial raises doubts about its efficacy 1, 2. Outcomes after ASCT are better in patients who achieve complete hematologic response after the ASCT3. One report has shown improved outcomes with combining one dose of the proteasome inhibitor bortezomib with high dose melphalan as part of conditioning regimen 4. Preliminary data from a recent study suggest that the outcome of treating AL amyloidosis with two cycles of bortezomib and dexamethasone followed by ASCT was superior to the outcome of the ASCT alone5. We describe our experience with giving 4-6 cycles of bortezomib and dexamethasone induction prior to high dose melphalan and ASCT in patients with systemic AL amyloidosis. Patients and methods: We included all patients who underwent autologous transplant for symptomatic systemic AL amyloidosis at our institution from October 2010 till June 2014. Five patients were included in the analysis and patient characteristics are described in table 1. All patient received 4 -6 cycles of induction with bortezomib and dexamethasone followed by autologous stem cell transplant using high dose melphalan (200 mg/m2). One patient also received six cycles of lenalidomide and dexamethasone prior to bortezomib based induction for lack of response. Hematologic and organ response were assessed using the definitions from the 10th International symposium on Amyloid and Amyloidosis. Overall survival was calculated by Kaplan Meyer’s method using Graphpad Prism 6.0 software. Results: There was no transplant related mortality. After median follow up of 13 months (12-25 months) all patient are alive. Toxicities from the ASCT were mostly cytopenias in the immediate post-transplant period which were managed as per the standard of care. Two patients achieved hematological complete response while one more had very good partial response and other two achieved partial response. Of the four patients with nephrotic range proteinuria, two patients had > 95% reduction in proteinuria, one had > 75% reduction in proteinuria and another patient had > 50% reduction in proteinuria. One patient had Liver involvement with elevated alkaline phosphatase which normalized post-transplant (table 2). The responses were maintained on last follow up and none of the patient had hematological or organ relapses. Discussion: Bortezomib alone and in combination with steroids has shown efficacy in AL amyloidosis, but its role in induction prior to high dose melphalan/ASCT to help achieve deeper hematological response is unknown. Our experience shows that this combination may be highly efficacious without significant toxicity. Limitations of our study include the small number of patients and absence of any patients with cardiac involvement, which is a worse prognostic marker. We conclude that the bortezomib and dexamethasone induction followed by high dose melphalan/ASCT for AL amyloidosis should be studied in prospective trials. Table 1.Patient Characteristics n=5Age, years 51.2 (44-62)Race (Caucasian)4 (80%)Gender ( female)3 (60%)Cardiac involvement 0 (0)Renal involvement 4 (80%)Serum creatinine ≥ 2.5 0 (0)Organ involvement ≥21 (20%)BM plasma cells > 10%1 (20%)Hgb ≤ 10 g/dl0 (0)LVEF Table 2. Outcomes n=5 Baseline After ASCT Hematologic response n=5 M protein 0.772 gm/dl 0.096 gm/dl 2 CR, 1 VGPR, 2 PR Renal response n=4 24 hours proteinuria 3.13 gm 0.432 gm 2 > 95% reduction, 1 >75% reduction, 1 >50 % reduction. Liver response n=1 Alkaline phosphatase 700 IU/L 62 IU/L Disclosures No relevant conflicts of interest to declare.

Introduction Intensive treatment of acute myeloid leukemia (AML) is associated with severe pancytopenia. Thrombopoietic agents have so far failed to mitigate treatment-associated thrombocytopenia. Platelet factor 4 (PF4) is a potent negative regulator of megakaryopoeisis and its levels correlate with platelet transfusion requirements in children treated for acute lymphoblastic leukemia. Anti-PF4 antibodies diminish chemotherapy-induced thrombocytopenia in mice. ODSH (2-O, 3-O Desulfated Heparin), a heparin derivative with low anticoagulant activity, is a potent inhibitor of PF4, mitigates heparin-induced thrombocytopenia and, like other heparins, may inhibit the CXCL12/CXCR4 axis. The purpose of this pilot study was to obtain preliminary data on the effect of ODSH when combined with intensive AML treatment. Methods Adult patients with newly diagnosed untreated AML (excluding acute promyelocytic and acute megakaryoblastic leukemia) were enrolled. Induction therapy consisted of cytarabine (100 mg/m2 as a continuous 24-hour infusion on days 1 – 7) and idarubicin (12 mg/m2 by intravenous bolus on days 1 – 3). During induction cycles, ODSH was given as a bolus of 4 mg/kg on day 1 followed by a continuous infusion of 6 mg/kg/day on days 1 – 7. Patients 60 or older received further post-induction therapy off study. Patients younger than 60 received consolidation therapy with high dose cytarabine (3 g/m2 every 12 hours on days 1, 3, and 5) along with ODSH given as a bolus of 4 mg/kg on day 1 followed by a continuous infusion of 6 mg/kg/day on days 1 – 5. Patients received transfusion and antibiotic support per standard guidelines. Patients did not receive growth factor support during induction cycles. Primary endpoints were to determine the safety and tolerability of ODSH combined with intensive AML therapy, and to obtain preliminary data on the effect of ODSH on platelet count recovery. Secondary endpoints included obtaining preliminary data on the effect of ODSH on complete remission rate and tolerability of chemotherapy. Data presented here are for induction cycles. Data for consolidation cycles will be presented at the meeting. Results Ten patients were enrolled. Two patients did not complete the full induction cycle due to complications unrelated to ODSH therapy and are not included in the analysis. The median age of the 8 patients (3 women) who completed the full induction cycle and were analyzed was 55 (range: 22 – 74). There were no ODSH-associated adverse events. Based on cytogenetic, molecular, or antecedent hematologic disorder, 1, 5, and 2 patients fell into the better, intermediate, and poor risk categories, respectively. Day 14 bone marrow biopsies were obtained on all 8 patients and were aplastic without detectable leukemia in all. Seven out of 8 patients who completed induction had evidence of a morphologic complete remission upon count recovery using IWG criteria. One of the 7 patients in morphologic remission had evidence of minimal residual disease by flow cytometry and cytogenetics. It is noteworthy that the 2 patients who did not receive a full course of induction therapy (treatment discontinued on Day 3 and Day 5 of the induction cycle, respectively) achieved a complete remission upon count recovery using IWG criteria with evidence of minimal residual disease by molecular testing. All 8 patients who received a full course of induction therapy were evaluable for platelet count recovery. Among those patients, the median day to obtain a sustained platelet count above 20,000/ml without transfusion was day 20 (range: 16 – 22). Five of the 8 patients who received a full course of induction therapy were evaluable for neutrophil count recovery. Among those patients, the median day to a neutrophil count above 1,000/mL was day 23 (range: 21 – 27). Conclusion We conclude that ODSH is well tolerated when combined with aggressive therapy for the treatment of AML. Results from this pilot study show that ODSH may enhance count recovery and treatment efficacy. These results justify further study of this strategy in a randomized trial. Disclosures Kennedy: Cantex Pharmaceuticals: Equity Ownership. Bavisotto:Cantex Pharmaceuticals: Consultancy. Marcus:Cantex Pharmaceuticals: Employment, Equity Ownership. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Lone Star Thiotherapies: Equity Ownership; Cantex Pharmaceuticals: Research Funding.

We conducted an open-label phase 1 study exploring the feasibility, safety, and biologic activity of epigenetic priming with decitabine before standard induction chemotherapy in patients with less-than-favorable risk of acute myelogenous leukemia (AML). We directly compared the clinical and DNA-hypomethylating activity of decitabine delivered at 20 mg/m2 by either a 1-hour infusion (Arm A) or a continuous infusion (Arm B) for 3, 5, or 7 days before a single, standard induction with infusional cytarabine (100 mg/m2 for 7 days) and daunorubicin (60 mg/m2 × 3 doses). Toxicity was similar to that of standard induction chemotherapy alone. Although we did not identify a maximum tolerated dose, there was more gastro-intestinal toxicity with 7 days of decitabine priming. Decitabine induced DNA hypomethylation at all dose levels and there was a trend toward greater hypomethylation in CD34+ bone marrow cells when decitabine was delivered by a short pulse (Arm A). Twenty-seven subjects (90%) responded to therapy: 17 with complete remission (57%) and 10 with partial remission (33%). Of the patients with partial remission to protocol treatment, 8 achieved remission to their next therapy, bringing the overall complete remission rate to 83%. We conclude that epigenetic priming of intensive chemotherapy can be safely delivered in an attempt to improve response rates. This trial was registered at www.clinicaltrials.gov as NCT00538876.

Background High frequency mutations in both the colony-stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1) have recently been described in World Health Organization (WHO)-defined chronic neutrophilic leukemia (CNL) (NEJM 2013;368:1781; Leukemia. 2013. Prepublished on 2013/04/23). Response to treatment with ruxolitinib (10-15 mg twice-daily) was also described in one patient with CSF3RT618I mutation (NEJM 2013;368:1781). Primary cells from this patient were reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). In a report of 12 patients with WHO-defined CNL, CSF3RT618I was seen in 10 (83%) patients, four (40%) of whom also expressed SETBP1 mutations (Leukemia. 2013. Prepublished on 2013/04/23). In the current study, we investigated the clonal distribution of mutant CSF3R and SETBP1in a CNL patient expressing both mutations and describe our observations regarding the effect of JAK inhibitors, both in vitro and in vivo. Methods Under institutional review board approved protocol, peripheral blood and buccal cells were collected and density gradient centrifugation was applied to enrich for mononuclear and granulocyte cell fractions. Antibody-labeled magnetic bead separation was used to obtain CD3+ and CD34+ cell fractions. DNA sequencing was used to screen for CSF3R and SETBP1 mutations. Single colonies were obtained by plating mononuclear cells in duplicate with cytokine enriched methylcellulose with and without the addition of fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for both SETBP1 and CSF3Rmutations. Results The study patient was a 66-year-old lady with history of radiation therapy after lumpectomy for breast cancer in 1997. In October, 2012 the discovery of three synchronous lesions in the left breast necessitated mastectomy. White blood cell count (WBC) was approximately 13 x 10(9)/L at the time. Subsequently, her WBC gradually increased to 180 x 10(9)/L. Bone marrow examination on March 15, 2013 showed predominantly granulocytic proliferation with over 95% cellularity and no dysplastic features or monocytosis. Cytogenetic studies and mutation screening for JAK2V617F and BCR-ABL1were negative. A diagnosis of CNL was made and the patient was placed on hydroxyurea therapy. i) In vitro studies The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34+ myeloid progenitors; neither mutation was present in buccal or CD3+ cells. Colony forming unit (CFU) assay-derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I. Fedratinib and the JAK1 inhibitor revealed activity in suppressing colony formation by >50% at a drug concentration of ≥600 nM. Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations. However, CSF3R unmutated colonies were more likely to emerge than SETBP1unmutated colonies after in vitro drug exposure, which resulted in the appearance of occasional colonies that were negative for both mutations. ii) Treatment with ruxolitinib Ruxolitinib treatment at 10 mg twice-daily was started on 6/10/13, in addition to 500 mg/day of hydroxyurea. At the time, WBC was 86.5 x 10(9)/L, Hgb 11.8 g/dL and platelets 122 x 10(9)/L. On 6/17/13, WBC decreased to 43.9 and hydroxyurea was held. On 6/25/13, WBC increased to 89.6 x 10(9)/L and ruxolitinib was increased to 15 mg twice-daily. On 7/15/13, WBC had further increased to 190 x 10(9)/L and hydroxyurea was added to the treatment regimen at 500 mg/day. On 8/2/13, WBC was recorded at 91.3 x 10(9)/L. Conclusions In a double mutated CNL patient, we found CSF3R and SETBP1 mutations to be myeloid cell-restricted and co-expressed in erythroid and granulocytic cells; the latter antedated the former in order of acquisition. In vitro JAK inhibition had non-selective activity in suppressing myeloid colony formation. Treatment of the study patient with single agent ruxolitinib was ineffective. Disclosures: No relevant conflicts of interest to declare.

Abstract 3578 Decitabine (DAC) is successfully used for treatment of patients (pts) with myelodysplastic syndromes and AML. Following cellular uptake, DAC is thought to be activated to DAC-TP and incorporated into DNA. The DAC-TP/DNA complex binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately anti-leukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated pts has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a sensitive and specific LC-MS/MS method for quantification of DAC-TP. The assay exhibited excellent accuracy and precision. The accuracy values were 83.7–109.4%, as determined by calculating the percentage of measured DAC-TP relative to the respective nominal concentrations (50, 500 and 5,000 nM) of the quality control samples. The within-day coefficients of variation (CVs) were 19.9 % (n=6) at 50 nM and 4.7–7.0 % between 500–5,000 nM; the between-day CVs (n=3) were 15.2 % at 50 nM and 7.5–10.2 % between 500–5,000 nM. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant MV4–11, and in THP-1 and FDC-P1/Kitmut cells (in vitro); and in bone marrow (BM) and spleen of normal and FDC-P1/Kitmut-driven AML mice (in vivo). DAC-TP reached peak levels (0.8, 1.4 and 0.5 pmol/106 cells) in 1–4 hours and declined to 20 % of its peak concentration after 24 hours incubation with 2.5 μM DAC in MV4–11, THP-1 and FDC-P1/Kitmut cells, respectively. Inhibition of hENT1 that mediates DAC transport into the cells and dCK that phosphorylates DAC into DAC-TP by NBTI and 2-thio-2′-deoxycytidine, respectively, significantly inhibited DAC-TP accumulation in AML cells. DAC-TP decay was instead blocked by tetrahydrouridine (THU)-induced inhibition of CDA, the catabolizing enzyme for cytidine and deoxycytidine and analogs. Consistent with these results, low dCK and hENTs but not CDA expression were detected in DAC-resistant MV4–11 cells, which showed 60 % decrease in DAC-TP levels as compared to their parental counterparts. DAC/DAC-TP-mediated downregulation of DNMT proteins (preferentially DNMT1 and DNMT3a) was also demonstrated in the AML cells even at DAC-TP concentrations as low as 0.1–1.3 pmol/106 cells in vitro after 4 hours DAC incubation. In the in vivo experiments, DAC-TP levels in leukemic mice were comparable to that in normal C57BL/6 mice, 0.3 pmol/106 cells in BM and 199.2 pmol/g tissue in spleen at 4-hours and 0.2 pmol/106 cells in BM and 165.3 pmol/g tissue in spleen at 24-hours following an i.v. bolus of 6.5 mg/kg DAC. In BM of leukemic mice, not only DNMT1 and DNMT3a but also DNMT3b protein expression reduced 80 % (DNMT3a) or diminished (DNMT1 and DNMT3b). The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells (PBMC) from AML pts treated with a 10-day regimen of DAC given 20 mg/m2/day i.v. over 1 hour. In BM samples, the mean DAC-TP levels were 0.8 ± 0.6 (Day 1) and 0.9 ± 0.5 pmol/106 cells (Day∼5) in complete responsive (CR) pts (n=4); and 0.4 ± 0.3 (Day 1) and 0.12 ± 0.02 pmol/106 cells (Day∼5) in non-responsive (NR) pts (n=3). In PBMC samples, the mean DAC-TP levels were 0.5 ± 0.2 (Day 1) and 1.2 ± 0.4 pmol/106 cells (Day∼5) in CR pts (n=3); and 0.02 ± 0.02 (Day 1) and 0.21 ± 0.04 pmol/106 cells (Day∼5) in NR pts (n=3). These data suggested that higher levels are seemingly associated with clinical response, but a larger number of pts need to be tested. In conclusion, monitoring the intracellular concentration of DAC-TP is feasible, and DAC-TP levels correlate with DNMT downregulation and may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens. Disclosures: No relevant conflicts of interest to declare.

The outcome of the majority of older AML patients (pts) remains poor despite advances made in both pathogenesis and prognostication. Epigenetic gene silencing by DNA hypermethylation contributes to AML pathogenesis. Recently we reported promising results in older previously untreated AML pts receiving low-dose of hypomethylating agent DAC alone. However these results can be improved upon. In our study pts with higher miR-29b expression were more likely to respond to DAC therapy (P=.02). MiR-29b directly or indirectly targets and downregulates the DNA methyltransferases (i.e. DNMT1, DNMT3A and DNMT3B) and has hypomethylating and tumor suppressor activity in AML. We recently showed that miR-29b is repressed by a Sp1/NFkB/HDAC1 complex, and that the HDACI AR42 can increase miR-29b expression by disrupting this complex in AML. Thus we hypothesized that priming AML cells with AR42 would result in miR-29b upregulation and increased response to DAC in AML pts. Here we showed first that in three AML cell lines i.e. THP-1, NB4 & Kasumi-1, miR-29b is upregulated (1.7, 2.4 & 1.6, fold respectively) as early as 1h after 1 µM AR42 vs DMSO control treatment. Confirming the upregulation of miR-29b by AR42, we next tested if this would sensitize the cells to DAC treatment. Priming THP-1 cells with 1 µM AR42 (for 12h) followed by 2.5 µM DAC treatment (for 24h) decreased the cell viability (measured by MTS assays) by 7-fold when compared to cells that were treated with the reverse drug sequence i.e., 2.5 µM DAC (for 12h) followed by 1 µM AR42 (for 24h). The decrease of cell viability was also higher than for each drug alone; 1 µM AR42 (for 12h) followed by 2.5 µM DAC treatment (for 24h) decreased the cell viability by 5-fold compared to 1 µM AR42 alone (for 36h) and 2-fold compared to 2.5 µM DAC therapy alone (for 36h). To test this effect in vivo we utilized an AML model constituted by FDCP1 cells expressing mutant KIT (KITmut) that we engrafted in NOD/SCID mice. Mice developed AML at 4 weeks from inoculation. The sequential administration of AR42 followed by DAC resulted in longer overall survival (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 119. doi:1538-7445.AM2012-119