Early treatment of CLL/SLL does not impact survival-reflecting limitations in detecting progression early and identifying asymptomatic patients likely to benefit from early treatment. Improved understanding of CLL/SLL biology would identify better prognostic/predictive markers. This study attempts to address these issues by determining the relationship between cytokine aberrations and poor clinical outcomes in CLL/SLL in the context of a genetic-based prognostic model. Fifty-nine serum cytokines/chemokines were measured in 28 untreated CLL/SLL patients. Patients were stratified as GR or int/PR using cytogenetics. Comparison of CLL/SLL with 28 HCs revealed increased expression of Th2 cytokines (IL-10, IL-5, sIL-2Rα; P≤0.01) and decreased levels of Th1 cytokines (IL-17, IL-23, IFN-γ; P≤0.003). In a multivariate analysis of GR versus int/PR groups, differential expression of sIL-2Rα maintained significance with increased expression in int/PR CLL/SLL. With median follow-up of 54.3 months after diagnosis, four patients incurred disease progression, with an IL-17/sIL-2Rα model predicting need for treatment in all cases. In summary, specific cytokine signatures are associated with genetically defined aggressive disease and predict need for therapy. This suggests utility in detecting disease progression early, identifying those likely to incur a survival advantage with early treatment, and directing future therapy.

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Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated the in vitro efficacy of the active form of novel agent sapacitabine, CNDAC, compared to current chemotherapeutic drugs Ara-C and mitoxantrone using two AML cell lines, HL-60 (promyelocytic) and THP-1 (monocytic), as well as bone marrow (BM) and peripheral blood (PB) cells collected from AML patients. Cell lines were exposed to compound for 3–6 days and primary cells for 4 days. The viability of primary cells was additionally evaluated 3, 7, and 31 days after removal of tested compound to determine the durability of the response. Our studies indicate that CNDAC and mitoxantrone have a greater impact on viability than ara-C in primary AML cells and AML cell lines. CNDAC is more effective at reducing viability and inducing apoptosis than ara-C at equivalent concentrations in the THP-1 cell line, which is defined as displaying resistance to ara-C. As sapacitabine has shown in vivo activity at clinically achievable doses, future studies are warranted to assess the potential for combining it with ara-C and/or mitoxantrone, with an emphasis on cells and patients insensitive to ara-C treatment.

2646 Introduction: Chronic lymphocytic leukemia (CLL)/Small lymphocytic leukemia (SLL) is one of the most common forms of indolent lymphomas in elderly adults. Currently, CLL is not treated until it develops into later stage disease. An increase in the knowledge of the biology of CLL could aid in the development of new treatment strategies for early stage CLL, thus positively impacting disease progression. We therefore investigated the cytokine levels present in peripheral blood (PB) serum of CLL patients and compared them to levels in healthy donors. Methods: PB was obtained from 25 untreated CLL and 3 untreated SLL patients and 29 normal healthy donors under an IRB approved protocol. Serum was stored at −80°C until analyzed. 86% of the patients were Rai 0/1, 3.5% Rai stage 2, and 10.5% unknown at diagnosis. Risk stratification based on cytogenetics and FISH found of the enrolled patients, 47% good, 25% intermediate, and 14% poor risk. The risk factor was unknown in 14%. β-2 microglobulin and 54 cytokine protein levels in 2 different serum samples per patient (collected at least one year post diagnosis) were measured in duplicate using MILLIPLEX™, a multiplex Luminex based technology. Genespring 11.5.1 software was used to convert the data into base-2 logarithmic values and then apply a median baseline transformation across all samples. Data is grouped according to the source of the sample (CLL or normal PB serum). A Filter on Volcano Plot analysis is then done. This filter analysis performs an unpaired t-test, which yields a p-value as well as computes the fold change of each cytokine. Results: As expected, β-2 microglobulin was significantly higher in CLL patients compared to normal samples (p=5.17×10−7, 1.79 fold). Using a minimum of a 1.5 fold change and p-value≤0.05, 16 cytokines had significantly higher expression and 9 cytokines had significantly lower expression in CLL samples compared to normal samples (see Table 1). Conclusion: These changes in cytokine levels help provide insight to the peripheral blood microenvironment, in which circulating CLL cells reside. Some cytokines were substantially higher in CLL patients; soluble IL-2 receptor alpha (sIL-2Rα) and stem cell factor (SCF). The substantially higher levels of sIL-2Rα have also been observed in follicular lymphoma (FL) and appear to predict which FL patients will have a reduced survival (Yang et al., Blood 2011). Levels of sIL-2Rα correlating to survival may be a phenomenon seen in all B-cell lymphomas. SCF is thought to be important in early B-cell development but its receptor c-kit (CD117) has not been reported to be expressed on B-CLL cells. The chemokine receptors CXCR4, CXCR5 and CCR7 are expressed on B-CLL cells. Interestingly, their corresponding ligands, CXCL12, CXCL13, and CCL21 are significantly higher (Table 1) in the PB serum of CLL patients. It is likely that existing immunomodulatory therapies, such as IMiDs, lenalidomide or thalidomide alter the PB levels of the cytokine milieau. Other successful hematological malignancy therapies involve targeted monoclonal antibodies. Therefore, a deeper understanding of the cytokine microenvironment will provide guidance in the development of future targeted therapies or highlight current therapies for other malignancies that could be promising for CLL. Disclosures: No relevant conflicts of interest to declare.

372 A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequent immune reconstitution during hematopoietic stem cell transplantation (HSCT) in the clinical setting. We have previously reported that suppression of the peptidase CD26/DPPIV (dipeptidylpeptidase IV) on mouse HSC/HPC as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al. Science 2004. 305:1000-3). We have also described that inhibition of CD26 on CD34+ or lin- cord blood (CB) cells improves engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007. 16:355-60). However, it has been reported that inhibition of CD26 on CD34+ G-CSF mobilized peripheral blood (PB) cells does not improve migration in response to SDF-1 or engraftment into NOD/SCID mice (Kawai K, et al, et al. Stem Cells and Dev 2007. 16:361-70). Given the widespread usage of un-fractionated mobilized PB as a source of cells for transplantation in both the autologous and allogeneic setting, the implications of also studying un-fractionated mobilized PB should not to be overlooked. We therefore investigated whether inhibition of CD26 activity on mobilized PB (mobPB) mononuclear cells (MNC) would have an effect on CXCL12/SDF-1 mediated adhesion and migration. These results were compared directly with data from CB MNC. Non-adherent MNCs from mobPB and CB were obtained by density centrifugation followed by adhesion to plastic for 90 minutes. By flow cytometry, CD26 expression was approximately 48% and 20% of the CD45+ cell population with Mean Fluorescent Intensity (MFI) of 1,200 and 1,000 for mobPB and CB MNCs respectively. Expression of CD26 was confirmed by Western Blot in total cell lysates from mobPB and CB MNCs. CD26 activity, as determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA), was 36.7±2.8 pmol pNA/minute for mobPB MNCs and 30.9±2.8 pmol pNA/minute for CB MNCs (n=16). Prior to use in functional in vitro assays, cells were first cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA) for 15 minutes in DPBS+0.2%BSA at 37oC, 5% CO2. To perform static adhesion assay, cells were treated with SDF-1α (0-800 ng/mL) for 30 minutes, loaded onto human fibronectin coated plates, and then allowed to incubate an additional 30 minutes at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB MNC was observed to result in an increase in adhesion as compared to untreated cells. Specifically, the percent adherence to fibronectin in response to 400ng/mL SDF-1α increased from 50.1±4.5% for untreated to 70.1±2.3% for DpA treated mobPB MNCs (p≤0.05, n=6). The percent adherence for CB MNCs increased from 48.2%±5.1 for untreated to 65.4±5.9% for DpA treated cells in response to 400ng/mL SDF-1α (p≤0.05, n=6). To assess migration, chemotaxis assays were performed by loading 2×105 cells into the upper chamber of a transwell plate, adding SDF-1α (0-400 ng/mL) in the lower chamber, and incubating for 2 hours at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB cells was observed to result in an increased migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 20.0±1.7% and 27.8±1.9% for untreated and DpA treated mobPB MNCs respectively (p≤0.01, n=7). The percent migration in response to 400ng/mL SDF-1α was 58.8±1.4% for untreated CB MNCs and 70.6 ± 2.9% for DpA treated CB MNCs (p≤0.01, n=4). These data indicate that a substantial portion of G-CSF mobPB cells express CD26 and exhibit CD26 peptidase activity similar to that seen on CB cells. In addition, CD26 inhibition on these cells results in an increase in functional response to SDF-1 as quantitated by adhesion and migration. Taken in the context of other studies examining sorted CD34+ mobPB our data suggest that un-fractionated mobPB may have inherently different regulatory properties due to the presence of accessory cells. Additionally, our data suggest that suppression of functional response of HSC/HPC to the chemokines SDF-1 by CD26 activity may be an endogenous regulatory mechanism that extends beyond CB to also include mobPB. Future studies are needed to pursue whether inhibition of CD26 activity can serve as a technique by which the trafficking of hematopoietic stem and progenitor cells from un-fractionated mobilized peripheral blood can be manipulated for possible clinical benefit. Disclosures: No relevant conflicts of interest to declare.

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3714 Hematopoietic stem and progenitor cell (HSC/HPC) trafficking into the bone marrow during hematopoietic stem cell transplantation (HSCT) is thought to be an important biological step that can be targeted to improve transplant efficiency and subsequent immune reconstitution. It has been reported that loss/inhibition of the peptidase CD26, also known as DPPIV (dipeptidylpeptidase IV), increases the transplant efficiency of mouse HSC/HPC (Christopherson, KW 2nd, et al. Science 2004). In a similar manner, inhibition of CD26 on CD34+ or lin− cord blood (CB) cells improves in vitro cell migration and in vivo engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007). There is currently controversy as to whether inhibition of CD26 on mobilized peripheral blood (mobPB) has benefit since it has been reported that inhibition of CD26 does not improve migration of G-CSF mobPB CD34+ cells in response to SDF-1 (Kawai K, et al. Stem Cells and Dev 2007. 16:361–70) but does improve the migration of G-CSF mobPB mononuclear cells (MNC) (Frank R, et al. ASH Abstract #372, 2009). Additionally, it has been reported that AMD3100 mobPB c-kit+ mouse cells have high CD26 expression similar to BM in contrast to G-CSF mobPB c-kit+ mouse cells which have very little CD26 expression (Bonig H, Exp Hem, 2009). The combination G-CSF + AMD3100 (a CXCR4 antagonist also known as plerixafor or Mozobil ®) has been recently introduced into the clinic for use in obtaining mobPB from Multiple Myeloma patients. We therefore investigated whether CD26 on human mobPB MNC obtained from Multiple Myeloma autografts mobilized with either G-CSF or G-CSF + plerixafor would have an effect on SDF-1 induced cell migration. To do this, non-adherent MNC from mobPB were obtained from discarded bags following the completion of stem cell infusion of previously frozen autografts. CD26 expression was detected by flow cytometery. CD26 activity was determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA). To assess migration, chemotaxis assays were performed by loading 5×104 cells into the upper chamber of a 96 well transwell plate, adding human SDF-1α (0–400 ng/mL) in the lower chamber, and incubating for 2 hours at 37°C, 5% CO2. By flow cytometry, CD26 expression was 21.61±2.19% and 21.65±3.68% of the CD45+ cell population for G-CSF and G-CSF + plerixafor mobPB MNCs respectively. CD26 activity was 15.4±1.2 pmol pNA/minute and 16.3±0.4 pmol pNA/minute for G-CSF and G-CSF + AMD3100 MNCs. During functional in vitro assays, cells were cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA). DpA treatment of G-CSF + plerixafor mobPB resulted in a significant increase in cell migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 21.3±2.4% and 31.6±3.0% for untreated and DpA treated G-CSF + plerixafor mobPB cells respectively (p=<0.01, n=10). The percent migration in response to 400ng/mL SDF-1α was 19.8±2.0% and 25.3±2.6% for untreated and DpA treated G-CSF mobPB cells respectively (p=0.21, n=7). These data indicate that CD26 inhibition, by treatment with DpA, results in a preferential increase in SDF-1 induced migration of G-CSF + plerixafor mobPB cells as compared to G-CSF mobPB cells. The preferential response does not appear to correlate to any change in CD26 expression or activity since both types of mobPB have equivalent expression and activity prior to the migration assay. Our data suggest that un-fractionated mobPB collected following mobilization using the combination of G-CSF + plerixafor (AMD3100) may have inherently different cell trafficking regulatory properties when compared to mobPB collected following G-CSF alone and that additional regulatory mechanisms allow the G-CSF + plerixafor mobPB to have an enhanced response to SDF-1 in the absence of CD26 suppression. Additionally, our data suggest that inhibition of CD26 peptidase activity may be more clinically relevant in the G-CSF + plerixafor mobilized patient population with regard to achieving improved stem cell trafficking. Disclosures: Fung: Genzyme: Honoraria, Speakers Bureau.