ALBERT

Description: Protein Z (PZ) is a vitamin K-dependent protein, whereas PZ-dependent protease inhibitor (ZPI) is a member of the serine protease inhibitor superfamily. ZPI rapidly inhibits factor Xa in the presence of PZ, calcium, and phospholipids and inhibits factor XIa in a PZ-independent fashion (Blood2000; 96:3049–3055). PZ circulates as a complex with ZPI in plasma and deletion of either the PZ or the ZPI gene is associated with the prothrombotic phenotype in mice. In addition, W303X or R67X nonsense mutations in the ZPI gene are reportedly associated with deep venous thrombosis in certain human populations. Western blot analysis of platelets stimulated with thrombin (0–200 mU/mL) showed they contained and released ZPI (approximately 200 ng/109 platelets) with the same molecular weight as plasma ZPI (72 kDa). The majority of the ZPI was released within 1 min. by 25 mU/mL thrombin. PZ was not detected in platelets by western blot analysis. Immunohistochemical staining using a monoclonal anti-ZPI antibody demonstrated a cytoplasmic fine granular staining pattern in maturing megakaryocytes in bone marrow aspirates and in circulating platelets, suggesting that ZPI may be stored in alpha granules. ZPI mRNA, however, was not detected by reverse transcriptase polymerase chain reaction (RT-PCR) in platelets or bone marrow aspirates, but was detected in human liver cDNA. RT-PCR for platelet factor 4 and glyceraldehyde 3-phosphate dehydrogenase mRNA showed amplified products with expected sizes. In conclusion, thrombin-releasable ZPI, but not PZ, is present in platelets and is most likely derived from the uptake of ZPI from plasma. ZPI released from activated platelets may play a role in the regulation of local coagulation at a site of injury.

Description: Abstract 2616 Background: Previous studies have been conflicting in their findings regarding whether NPM1 gene mutations may be a marker for response to all-trans retinoic acid (ATRA) given as an adjunct to intensive chemotherapy in patients with acute myeloid leukemia (AML) (Schlenk RF, Haematologica, 2009, 94, 54–60, Burnett AK, Blood, 2010, 115,948, and Schlenk RF, abstract #80, ASH 2011). Patients and Methods: we examined the impact of the addition of ATRA among patients with diploid cytogenetics treated on a randomized phase II study of fludarabine + cytarabine + idarubicine +/− GCSF +/− ATRA with available data on their NPM1 mutation status. Between September 1995 and November 1997, 215 patients with newly diagnosed AML (n=153) or high-risk MDS (RAEB or RAEB-T; n=62) were enrolled in the study. They had to have one of the unfavorable features of age 〉 71 years, antecedent hematological disorder (AHD), therapy- related disease, or high bilirubin (〉2.9 mg/ml) or creatinine (〉1.5 mg/ml) to be eligible to participate in the study (Estey E, Blood, 1999, 93, 2478). Among them 70 patients had diploid cytogenetic and are the subjects of this analysis. Results: The median age of the 70 patients was 66.5 (range, 23–87), 60% had AHD, 15.7% had therapy-related disease, and 40% were older than 71. Bone marrow samples were available for all patients for examination by immunohistochemistry (IHC) for the presence of cytoplasmic NPM (correlating to NPM1 mutation). Twenty (29%) of patients had NPM1 mutation. Among them 7 (35%) did and 13 (65%) did not receive ATRA in combination with chemotherapy. Complete remission (CR) was achieved in 5 (71.4%) of patients treated with ATRA as compared to 9 (69.2%) without ATRA (p=NS). The median event-free survival was 82 weeks vs. 56 weeks for patients receiving ATRA or not [(p=0.5), ranges, (10–173 weeks) and (1–646 weeks), respectively]. The median overall survival was 41 weeks vs. 60 weeks for patients receiving ATRA or not [(p=0.74), ranges, (3–452 weeks) and (0–735 weeks), respectively]. Conclusion: There was no significant difference in CR rate, EFS, or OS in this higher risk population of patients with NPM1 mutated diploid AML or high-risk MDS receiving ATRA as an adjunct to chemotherapy. Disclosures: Kantarjian: Genzyme: Research Funding. Ravandi:genzyme: Research Funding.

Description: Abstract 1703 Myelodysplastic syndromes (MDS) are a group of myeloid malignancies that originate from bone marrow hematopoietic stem/ progenitor cells (HSC). We have previously reported that deregulation of Toll-like Receptor (TLR) 2 mediated innate immunity signaling contributes to the pathogenesis of MDS through its impact on HSC. Several amyloid family peptides have been reported to be endogenous TLR2 agonists, including Ab-42 and the serum amyloid protein A (SAA1). To further dissect the role of this TLR2 centered molecular signaling in MDS, we characterized these amyloid peptides in MDS. Elisa assays indicated that the concentration of SAA1 protein in bone marrow plasma of MDS patients (N=17) was 3 fold higher (p-value 0.01) compared to normal controls, whereas in the same patient cohort the concentration of Ab-42 in MDS bone marrow plasma was elevated by ∼ 1.5 fold. We then examined expression of SAA1 in bone marrow CD34+ cells of MDS. RT-PCR results indicated that SAA1 RNA level was increased by 18 fold in MDS CD34+ cells compared to control (N=64, p-value 0.0001). While it is known that expression of SAA1 is highly sensitive to acute and chronic inflammation and can be regulated by multiple inflammatory cytokines such as IL-6 and IL-8, its regulation by TLR2 mediated signal in HSC is unknown. To examine the impact of TLR2 signal on SAA1 expression, we profiled RNA expression in an OCI-AML3 cell line that stably expresses the shRNA against TLR2. Microarray analysis indicated that in TLR2 shRNA expressing cells, RNA level of SAA1 decreased by ∼50%. Similar decrease of SAA1 RNA was also detected when primary CD34+ cells isolated from the bone marrow of MDS patients (N=4) were treated with retroviral mediated TLR2 shRNA. These results suggest that TLR2 deregulation may contribute to the overexpression SAA1 in MDS CD34+ cells. Further, we examined whether elevated bone marrow SAA1 level could affect HSC function. We treated in vitro cultured bone marrow CD34+ cells with a recombinant 105 amino acid SAA1 peptide and then analyzed its impact on hematopoietic colony formation in methylcellulose-medium. Results derived from four CD34+ HSC samples indicated that 1mg/ml SAA1 peptide led to a 16% decrease of hematopoietic colony numbers (p-value 0.02). This result suggests that SAA1 can negatively affect the hematopoietic capacity of HSC. Taken together, this study suggests that SAA1 may be part of the deregulated TLR2 mediated innate immunity signaling network that contributes to MDS pathogenesis. Previous studies have indicated that SAA level was elevated in HSC of the mice with telomere dysfunction (Sahin et al. Nature 2011). Therefore, we hypothesize that SAA is involved in the pathogenesis of MDS during ageing. Further molecular analysis of the role of SAA1, and its clinical impact (that is being analyzed), during ageing and MDS require further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1700 To better understand molecular bases of MDS pathogenesis, we performed a genome-wide CHIP-Seq analysis of H3K4me3, a histone mark associated with gene activation. In MDS CD34+ cells (N=4), 36 genes showed higher levels of promoter-H3K4me3 compared to controls. 10 of 11 randomly selected genes from these 36 showed increased mRNA expression in MDS CD34+ cells (N=100), supporting the positive correlation between expression and increased promoter H3K4me3. Pathway Analysis indicated that a majority of these genes are involved in innate immunity signal and NF-kB activation. This was validated by increased phospho-p65 in MDS bone marrow CD34+ specimens (N=15). Knock-down of 4 of these genes (C5AR1, FPR2, PTAFR and TYROBP) in OCI-AML3 cells resulted in reduction of p-p65. The observation of innate immunity signal activation and epigenetic deregulation led us screen key innate immunity activators, the Toll-like Receptor (TLR) family genes, and histone methylation regulators in MDS CD34+ cells. Among 8 TLRs and 24 histone methylation regulators, TLR1, 2 and 6, and Jmjc-domain histone demethylase JMJD3 were found to be significantly overexpressed in MDS CD34+ cells compared to control counterparts. This is of biological significance because TLR1 and 6 form functional hetero-dimmers with TLR2. Also, JMJD3 expression can be activated by TLR-NFkB in macrophages. To study TLR2 activation in HSC, we treated CD34+ cells with TLR2 agonists. This led to increased expression of JMJD3, supporting the biological function of TLR2 signal in HSC. We observed that JMJD3 knockdown in OCI-AML3 cells led to reduced expression of innate immunity genes (N=7), accompanied with changes of promoter H3K27me3 and H3K4me3, suggesting that JMJD3 forms a positive feedback to perpetuate innate immunity pathway. To further study the role of TLR-JMJD3 in MDS, we performed capture deep sequencing in 40 MDS bone marrow mononuclear cells (TLR1, 2, 4 and 6, JMJD3, UTX, UTY and JMJD1A). Seven different rare SNP in the coding regions of JMJD3, UTY, JMJD1A, and TLR2 were identified in 5 patients. Among them, one SNP of TLR2 causes a missense mutation, changing a conserved hydrophobic Phe217 to a hydrophilic Ser. We then analyzed the presence of TLR2 F217S in 150 MDS samples by Sanger sequencing. TLR2 F217S was observed in 17 (11%) patients. To evaluate the somatic nature of this alteration, CD3+ cells from 15 corresponding patients were sequenced and only two (13%) CD3+ cell samples carried TLR2 F217S. In the available 9 CD34+ cDNA samples, TLR2 F217S was observed in 8 (90%). We then expressed wild-type or F217S TLR2 in 293T cells, a cell line without endogenous TLR2 expression. Reporter assays indicated that in the absence of TLR2 agonist wildtype and F217S mutant TLR2 led to similar levels of NF-kB activation, whereas F217S led to an increased NF-kB activation compared to wildtype at the presence of TLR2 agonists. F217S also led to increased activation (phospho- and polyubiquitin-) of IRAK1, a key signal mediator for TLR signaling. These results suggest that TLR2 F217S led to more robust innate immunity signal activation when stimulated by agonists. We further studied the impact of TLR2 activation on hematopoietic differentiation. Colony formation of CD34+ cells indicated that TLR2 agonists led to decreased number of erythroid colonies, which was confirmed by flowcytometry that demonstrated TLR2 agonist treatments could reduce the number of CD71 high/HLADR low featured erythroid precursors. To examine the effect of targeting TLR2-JMJD3 in primary MDS cells, we transduced MDS CD34+ cells with shRNAs. In 4 CD34+ cases isolated from lower risk MDS, 3 transduced with JMJD3 shRNA and 4 transduced with TLR2 shRNA had increased ratio of erythroid colonies. In average, JMJD3 and TLR2 shRNA transduction led to a 50% increase in erythroid colonies. This was accompanied by increased expression of genes positively involved in differentiation of erythroid lineage (GYPA, GATA1 and EPOR). Finally, CD34+ cell mRNA expression levels of four genes in this study (NCF2, AQP9, MEFV and TLR1) were associated with overall survival of patients. Taken together, these studies highlight the implication of the deregulation of TLR2-JMJD3 mediated innate immunity signals in MDS pathogenesis and suggest that intervention of this pathway may have therapeutic potential in MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Background: MYC mRNA overexpression has been described in acute myeloid leukemia (AML), but no studies have assessed MYC protein expression in AML where its clinical significance is unknown. In this study our aim is to assess MYC protein expression across all AML subtypes and explore its prognostic value in in a subset of patients (pts) with particularly poor prognosis, namely those with AML with myelodysplastic syndrome (MDS)-related changes (AML-MRC) and therapy-related AML (t-AML). Characterized by unfavorable cytogenetics, the prognosis of patients with AML-MRC and t-AML is often dismal, even in patients who undergo allogeneic stem cell transplant. There is a need for further molecular characterization of AML-MRC and t-AML to identify prognostic markers to optimally risk-stratify to better guide treatment decisions in these pts. Objectives: In the current study we sought to: (1) assess MYC protein expression by immunohistochemistry (IHC) performed on bone marrow (BM) specimens of pts with all types of AML, including acute promyelocytic leukemia (APL), and myelodysplastic syndromes (MDS), and (2) explore the prognostic significance of MYC protein expression in AML-MRC and t-AML (2008 WHO criteria). Methods: MYC protein expression was assessed by IHC on BM obtained from pts with AML, APL, and MDS, and from patients without hematologic neoplasms (normal controls). MYC expression was considered positive if 〉 5% blasts in BM showed nuclear reactivity. For cases classified as t-AML and AML-MRC by WHO 2008, MYC expression was correlated with molecular, cytogenetic, and clinical outcome data. X-tile software was used to identify the optimal cutoff point to dichotomize pts by MYC protein expression. Results: We evaluated BM MYC expression in 306 pts during 2006-2013 with newly diagnosed AML (n=246) or APL (n=11), previously treated AML (induction failure or relapse) (n=30), MDS (n=19), AML-MRC (n=68), t-AML (n=10), and normal BM (n=11). The median age was 61 yrs (13-88) with 54% men. Normal BM showed negligible MYC expression, ≤2% positive nuclei. The median MYC expression varied across diseases: newly diagnosed AML 25% (range 0-38%), APL 50% (range 19-75%), previously treated AML 20% (range 0-80), MDS 5% (range 0-30%) (differences between groups, p=0.0001). High MYC expression correlated with high LDH (rs = 0.285, p21% MYC-positive blasts have an inferior OS, DFS, and remission rate than pts with ≤ 21% MYC-positive blasts. Increased MYC protein expression is independent of rearrangement status by FISH. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 291 Although cytogenetic abnormalities are common in MDS, search for genetic alterations has been less informative with few prevalent abnormalities thus far known. To identify genes aberrantly activated in MDS, we developed a novel approach based on chromatin immuno-precipitation combined with massive parallel sequencing (CHIP-Seq) using the Solexa 1G sequencing technology. To our knowledge this is the first example of the use of this technology in primary human samples. For CHIP analysis we used an antibody against H3K4me3 (histone-H3-lysine 4-trimethylation). H3K4me3 is a chromatin mark of gene activation that localizes to active gene promoter regions. CHIP-Seq was performed in CD34+, CD34 neg cells and whole bone marrow (WBM) from 6 patients with MDS and 4 normal controls. In total 30 samples were sequenced. Patients samples were obtained at the time of initial referral at MDACC and were sorted immediately using standard separation procedures. When compared to normal controls for each cellular compartment, we identified 36, 156 and 32 potential active gene promoters associated with H3K4me3 in CD34+, CD34 neg cells and WBM respectively. Of importance, gene promoter regions identified did not overlap among the different cellular compartments analyzed (differences were observed comparing normal vs MDS but also among different MDS compartments), indicating that chromatin structure and gene expression profiles are aberrant and distinct in non-CD34+ cells that may also contribute to the pathobiology of MDS. Here we focus on H3K4me3-associated gene promoters in CD34+ cells. To confirm the results obtained with the CHIP-seq approach, we studied the expression levels of the top 9 CHIP-Seq identified genes in an independent cohort of in CD34+ cells obtained from 54 MDS at the time of initial diagnosis. Patient characteristics have been previously reported (Leukemia, in press): 11 (20%) low risk, 20 (37%) int-1, 15 (27%) int-2 and 8 (14%) high risk by IPSS. We confirmed gene expression up-regulation of 7 (C5AR1, FPR1, FPR2, AQ9, FYB, FCAR, IL8RA) of 9 genes detected by CHIP-Seq. Using Ingenuity Pathway Analysis of the 36 genes identified in CD34+ cells revealed NF-κB as central activated knot in CD34+ cells. This was confirmed by phospho-p65 immuno-staining in primary cells. Furthermore up-regulation of all 10 NF-κB activation associated genes was confirmed in MDS CD34+ cells by Q-RT-PCR. Transfection of OCI-AML3 cells with a siRNAs cocktail targeting 4 of the CD34+ NF-κB activation genes dramatically repressed NF-κB activation as well as expression and promoter NF-κB association of JMJD3 gene, a known NF-κB transcriptional target. JMJD3 encodes a Jmjc-domain K27me3 demethylase, which positively regulates H3K4me3. We further characterized expression levels of 17 known histone demethylases known in human in 35 patients with MDS and identified JMJD3 as the only histone demethylase overexpressed in MDS CD34+ cells. siRNA targeting JMJD3 reduced expression and promoter H3K4me3 levels of several CHIP-Seq detected MDS- CD34+-NF-κB activation genes. Finally expression profile of JMJD3 and the panel CD34+-NF-κB activation genes in the 54 patients with MDS indicated that expression levels were consistently overexpressed in patients with higher-risk (high and int-2) disease compared to patients with lower (low and int-1) risk disease. In view of the known antiapoptotic and proliferative role of the NF-κB pathway, this data indicates that expression of upstream and downstream modulators of NF-κB signaling, regulated at the chromatin level by JMJD3, have a role in MDS progression and could serve as therapeutic targets. Through this novel in vivo CHIP-Seq analysis, we demonstrated that a positive regulatory loop exists in MDS CD34+ cells. This loop contains JMJD3 promoted gene activation through positive regulation of H3K4me3, which leads to NF-κB signaling activation, and then further promotion of JMJD3 expression and activation of the whole signaling cascade. Our study also demonstrates that in vivo CHIP-Seq can be used to discover disease specific targets. Disclosures: No relevant conflicts of interest to declare.

Description: The pathogenesis of MDS is multifactorial including both cell intrinsic alterations, such as mutations, and cell extrinsic stimuli such as immune deregulation. The prognosis of patients that lose response to hypomethylating agents (HMAs) is very poor and the mechanisms are not understood. Leukemia cells develop multiple resistance mechanisms to escape host immune response. Programmed death-1 (PD-1) is a negative costimulatory receptor on activated T lymphocytes. Expression of PD-1 ligands on tumor cells can induce immunosuppressive T-cells. Recent studies have indicated that demethylation of PD-1 leads to exhausted CD8+ T cells after chronic virus infection. We hypothesized that dysregulated PD-1 immune checkpoint signaling may be involved in pathogenesis of MDS and resistance to HMAs. We first evaluated the mRNA expression levels of PD-L1, PD-L2, PD-1 and CTLA4 by Q-PCR in bone marrow CD34+ cells from 124 patients which include 69 with MDS, 46 with CMML and 9 with AML. Seventy two (58%) patients were previously untreated. We observed aberrant up-regulated mRNA expression of PD-L1 in 39 patients (34% with fold 0-843.3), PD-L2 in 17 patients (14% with fold 0-22.5), PD-1 in 18 patients (15% with fold 0-76.7) and CTLA4 in 10 patients (8% with fold 0-25.1). No significant differences in gene expression were observed when comparing patients that had and had not received prior therapy. Statistically significant relationships were identified between elevated PD-1 expression and increased age (p=0.008), while elevated PD-L2 expression correlated significantly with female gender (p=0.005). Both elevated PD-L1 and CTLA4 expression correlated with MDS subtype, (p=0.034) and (p=0.012), respectively. Additionally, elevated CTLA4 expression correlated with higher white blood cell count (p=0.021), lack of prior therapy (p=0.02) and lower MDS IPSS score (p=0.027). We then performed an analysis of the impact on survival of the 4 gene expression in patients that had not received prior therapy. Patients with lower expression of PD-L1 had a non-significant trend towards better survival (31.5 months versus 16.2, p=0.24). Forty six of 124 patients analyzed received HMAs, and within these 46 patients lower PD-L1 expression correlated with a significantly improved overall response rate (67% vs 25%, p=0.038). Aberrant up-regulation of these 4 genes was also observed in peripheral blood mononuclear cell (PBMNC) from 61 MDS, CMML and AML patients. The relative expression of PD-L1 was significantly higher in MDS (p=0.018) and CMML (p=0.0128) compared to AML. Of interest, mRNA expression of these 4 genes was significantly higher in PBMNC than in CD34+ cells except PD-L1. By immunohistochemical (IHC) analysis, a strong correlation was observed between protein and mRNA expression. By IHC, we observed that leukemia blasts were positive for PD-L1 whereas stroma/non-blast cellular compartment was positive for PD-1 in bone marrow biopsies from MDS, CMML and AML patients. We then analyzed effect of HMAs on these four gene expression in cohort of 61 patients treated with different trials of epigenetic therapy. Treatment resulted in up regulated expression of PD-L1 in 57% of the patients (with maximum induction fold of 4.8), PD-L2 in 57% (maximum fold of 15.77), PD-1 in 58% (maximum fold of 50.26) and CTLA4 in 66% (maximum fold of 29.9). Of importance, patients resistant to therapy had increased gene expression compared to patients that achieved response (fold 5.3 vs 0.4 for PD-L1, 6.2 vs 0.4 for PD-L2, 3.0 vs 0.4 for PD-1 and 5.4 vs 0.7 for CTLA4). Comparing patients without and with expression induction, the median survival was 11.7 and 6.6 months (p=0.122) for PD-L1, 12.5 and 4.7 months (p=0.029) for PD-L2, indicating a better prognosis in patients without PD-L1 and PD-L2 induction treated with HMAs. To model this observation, we treated KG-1 and THP1 cells with different concentrations of decitabine (0 to 10 uM) and observed a dose dependent up-regulation of PD-L1 and PD-L2 in THP1 cells, and CTLA4, PD-1, PD-L1 in KG1 cells. Exposure to decitabine resulted in demethylation of PD-1 in these cell lines, and the demethylation effect was also observed in HMAs treated MDS and AML patients. This study suggests immune checkpoint PD-1/PD-1 ligands signaling may be involved in MDS pathogenesis and resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML. Fig.1 PD-L1 expression in MDS CD34+ cells by IHC. Fig.1. PD-L1 expression in MDS CD34+ cells by IHC. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points VCAM-1/VLA-4 triggers reciprocal NF-κB activation in leukemia and stromal cells and mediates cross-talk between leukemia and stromal cells. VCAM-1/VLA-4 and NF-κB signaling plays a pivotal role in the development of leukemia chemoresistance.

Description: Background Risk of VTE is high in cancer patients, especially, in patients with APC. Treatment with chemotherapy further increases the risk. Purpose of the study was to evaluate the safety and efficacy of primary thromboprophylaxis with dalteparin in reducing the incidence of VTE in APC patients planned to start chemotherapy; and to determine the baseline risk factors/biomarkers predictive of VTE. Methods Patients with metastatic or locally advanced pancreatic cancer planned to start chemotherapy were randomized 1:1 to dalteparin vs control arms, stratified for the presence of metastasis and central venous catheter (CVC). The treatment arm received dalteparin 5000 U SQ daily for 16 weeks during chemotherapy and the control arm received chemotherapy alone. Bilateral compression ultrasound of the lower extremities was performed at baseline, and during study (weeks-8 and-16). In addition, blood was collected to identify biomarkers such as, plasma D-dimer levels, platelet activation markers (P-selectin), thrombin-antithrombin complex (TAT), prothrombin fragments 1 and 2 (F1+2), and cytokine levels. Univariate and multivariate logistic regression analysis of clinical and laboratory parameters were done to identify risk factors associated with the development of VTE. Results Of 87 patients enrolled, 75 were randomized to dalteparin (38 patients) or control (37 patients) arms; 8 did not meet the eligibility criteria (including 6 found positive for incidental VTE on screening ultrasound), and 4 withdrew consents before randomization. There were 41 males and 34 females; with median age 52 (range, 36-77 years). Over half of the patients (55% dalteparin arm and 54% control arm) completed 16 weeks on study. All 75 patients were evaluable for response in an intent-to-treat analysis. During the study, the incidence of VTE was 22% [8/37 patients; 2 pulmonary emboli (PE) and 6 deep vein thrombosis (DVT)] on the control arm as compared to 5% (2/38 patients; both DVT) on the dalteparin arm (p = 0.02). In the multivariate analysis, baseline plasma levels of D-dimer, ECOG performance status, presence of CVC, and prophylaxis with dalteparin were independent factors predictive of risk for VTE, as shown below. There was no statistically significant difference in overall survival between the two arms; however, there were higher proportion of patients with elevated baseline D-dimer levels in the dalteparin arm than the control arm (≥ 5000 ng/mL 16% vs 3%). Elevated baseline D-dimer level (≥ 5000 ng/mL) was also predictive of the presence of silent or asymptomatic VTE at screening for study entry (p=0.001), suggesting its potential value in identifying patients with silent VTE. Treatment with dalteparin was well tolerated; the main adverse events included minimal bruising (5/34, 15%), or pain (2/34, 6%) at the injection sites. There were no clinically significant bleeding episodes in the dalteparin arm. Conclusions The results of this study showed that the incidence of VTE is very high in patients with APC. Primary thromboprophylaxis with dalteparin was well tolerated and was associated with 75% reduction in the incidence of VTE in ambulatory patients with locally advanced or metastatic cancer while receiving chemotherapy. Baseline risk factors such as elevated D-dimer levels may help identify high risk patients for primary thromboprophylaxis as well as patients with the presence of asymptomatic VTE. Disclosures: Vadhan-Raj: Eisai: Research Funding. Off Label Use: Fragmin (Dalteparin): Prophylaxis of VTE in ambulatory cancer patients while receiving chemotherapy.

Description: Abstract 3845 Introduction: Matrix metalloproteinases (MMPs) belong to a unique family of zinc dependent endopeptidases, they are strictly regulated by their endogenous inhibitors called tissue inhibitor of MMPs (TIMPs). MMPs have been associated with tumorigenesis. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in nonproteolytic manner. MMPs play a key role during invasion and metastasizing of cancer cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumors. Little is known about their role in MDS. A total of 23 different human MMPs and 4 TIMPs have been identified. To better understand the role of MMPs in MDS, we performed an expression profile screen study by QPCR to detect the expression level of all MMP and TIMP family members, except MMP23, in CD34+ cells from a cohort (N=10) of newly diagnosed, untreated patients with MDS, and compared the expression level with CD34+ cells from 5 normal donors. Of these 26 genes, we identified MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 as aberrantly up-regulated genes in MDS and expanded the study to a larger cohort of patients to correlate with clinical features and clinical outcomes. Methods: CD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donors were evaluated for MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 expression profiling. CD34+ cells were sorted from patients and normal donor bone marrow. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, all assays were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect MMP9 protein level in cytospin from CD34+ cells. MMP9 abtibody was obtained from R&D systems, MMP9 ELISA kit (R&D systems) was used to detect the MMP9 protein level in bone marrow plasma. Results: For the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By Cytogenetics, diploid 44 (45%), 20q- 7(7%), −5/5q- 7 (7%), −7/7q- 7 (7%), −5/5q- and −7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By QPCR and comparing MDS CD34+ cells with normal CD34+ cells, aberrant up-regulation (fold〉2) of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 was detected in 40%, 93.8%, 94%, 84%, 15.6%, 45%, 21% and 27% of patients respectively. Up-regulation of MMP8, MMP9 and MMP25 were significant with a mean fold value 350.7 (0–3363.7), 1112.4 (0–15641) and 143.8 (0–1017.9) respectively. We performed MMP9 immunohistochemistry of MDS CD34+ cytospins from 16 pts with higher and lower MMP9 RNA relative expression level and found consistent protein expression level in cytoplasm. No elevated MMP9 protein levels were detected in bone marrow plasma. We then performed an analysis of associations with clinical variables and observed that relative expression value of MMP9 was associated with lower bone marrow blast (p=0.001) and longer survival (p=0.02) (figure 1). We did not found association between survival and the other 7 up-regulated genes. Conclusions: Bone marrow CD34+ cells from patients with MDS have abnormal up-regulation of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3. MMP9 up-regulation is associated with longer survival. Our results suggest that MMP-9 could be a useful prognostic indicator for MDS and that this family of proteins needs further study in MDS. Disclosures: No relevant conflicts of interest to declare.