ALBERT

Description: Abstract 3737 Background: Hodgkin lymphoma (HL) is among the most curable lymphomas, however 20%-30% of patients relapse after initial chemotherapy, or have primary refractory disease. While 30–50% of these patients may be cured with second line chemotherapy and autologous stem cell transplant, patients who do not obtain a complete response (CR) prior to transplantation, or who relapse after second line therapy have few effective therapeutic options. Novel treatment strategies for these patients are needed. It has been previously shown that in patients with classical HL the malignant Hodgkin Reed-Sternberg cells (HRS) express a high level of the Interleukin-3 receptor (IL-3R). Therefore, we hypothesized that SL-401, a novel biologic conjugate consisting of IL-3 linked to diphtheria toxin, which targets IL-3R, may be an effective approach for selectively targeting and killing HRS cells. Methods: We first assessed the expression of the IL-3R α-chain (CD123) on two nodular sclerosing (HDLM-2 and L-428) and two mixed cellularity (KM:H2 and L-1236) HL cell lines by flow cytometry. Cells were washed with PBS and stained with CD123 PE (BD Pharmingen) as well as other antibodies, including CD15 FITC (BD Pharmingen), for 20 minutes at 4°C and again washed with PBS. Stained cells were acquired using the LSR II (BD) and data were analyzed using Flow Jo (Tree Star). We then tested the sensitivity of these cell lines to SL-401 using a CellTiter Glo in vitro cytotoxicity assay. A CD123 positive eyrthroleukemic cell line (TF-1/H-ras) with known sensitivity to SL-401 was used as a positive control. The cell lines were cultured in the presence or absence of SL-401 for 48 h and assessed for cell viability at concentrations ranging from 3×10−7 to 1.3 μg/ml. Results: We found high expression of CD123 on HDLM-2 and L-428 HL lines (99% and 89%, respectively) and low-to-moderate expression of the receptor on the L1236 and KM-H2 lines (19.5% and 1%, respectively). Based on CD123 expression, we assessed the sensitivity of these cell lines to SL-401. The L428 and HDLM2 cell lines, which exhibited a high expression of CD123, showed sensitivity to SL-401 relative to control starting at approximately 0.3 ng/ml. Cell viability was reduced to 64.8 ± 5% for L-428 and 68.2 ± 8% for HDLM2 when incubated at the highest concentration of drug (1 μg/ml (L428) and 0.7 μg/ml (HDLM2)). The low CD123 expressing cell lines L1236 and KM:H2 were less sensitive to SL-401 and did not exhibit a significant reduction in cell viability even at the highest concentration of SL-401 tested (1.3 ug/ml; 100 ± 2% and 88.4 ± 6%, respectively). Other lymphoid malignant cell lines with CD123 expression reported in the literature were also tested. Interestingly, the T Acute Lymphocytic Leukemia (T-ALL) cell lines DND41 and P12 showed marked sensitivity to SL-401, with a reduction in cell viability to 55.9 ± 4% and 47.9 ± 6%, respectively, in the presence of 1.3 μg/ml of drug. Conclusion: These results suggest that CD123 expression may vary as a function of HL histology, and that sensitivity to SL-401 may correlate with CD123 expression. Based on these results, SL-401, which is currently being evaluated in clinical trials of patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myeloid leukemia, may be a potential treatment strategy in refractory HL, and warrants further exploration in T-ALL. Exploration of CD123 expression in primary and relapsed/refractory patients with lymphoid malignancies as well as in vivo studies with SL-401 in this setting is currently underway. Disclosures: Brooks: Stemline Therapeutics, Inc: Employment, equity options. Cirrito:Stemline Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Bergstein:Stemline Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. O'Connor:Merck: Research Funding; Spectrum: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding.

Description: Abstract 4860 Background: Programmed death-1(PD-1) and programmed death-1 ligand (PD-L) signaling are involved in the functional impairment and “exhaustion” of T cells in conditions such as chronic viral infection and in tumor immune evasion. The interaction of PD-1 with its ligand PD-L suppresses T cell function. Up-regulation of PD-L has been demonstrated both in Hodgkin lymphoma (HL) cell lines, and in primary Hodgkin Reed Sternberg (HRS) cells. PD-1 is markedly elevated in the tumor infiltrating lymphocytes of HL patients, leading to the hypothesis that T cell exhaustion and deficient anti-tumor immunity induced by the activation of the PD-1-PD-L signaling pathway may play a key role in creating a permissive milieu for HL. In this setting, elevated PD-1 expression in HL patient T cells may be a biomarker of disease activity. We investigated this hypothesis by examining PD-1 expression in the peripheral T cells of patients with relapsed/ refractory HL. Methods: Patients with relapsed or refractory HL who enrolled in a clinical trial of an HDAC inhibitor in combination with Niacinamide between December of 2010 and July of 2011 were eligible to participate. PD-1 levels were assessed pre-treatment, and at selected timepoints during therapy. The levels of PD-1 expression for 5 relapsed/refractory HL patients and 4 healthy control subjects were evaluated by flow cytometry. An aliquot of cells (1 × 106/mL) was washed and stained with: CD3 APC-H7, CD8 PerCP-Cy5.5, CD4 FITC and PD-1-APC, for 20 minutes at 4°C. Dead cells were excluded using the Live/Dead Fixable Staining Kit (Invitrogen), and stained cells were acquired using the LSR II flow cytometer (BD). Compensation (parallel controls using cells singly stained for each color) and data analysis were performed using FlowJo flow cytometry analysis software (TreeStar). The percentage and/or mean fluorescence intensity (MFI) of PD-1+ cells within the live CD3+CD4+ and CD3+CD8+ populations was compared to isotype controls to establish baseline values and to normal control subjects. Results: The median age of the 5 HL patients was 32 (range 25–73). The median age of the healthy control subjects was 43 (range 39–49). Three of the HL subjects were male; all 4 of the normal controls were female. All 5 of the HL patients were heavily pretreated with an average number of prior regimens of 7.8 (range 5–13). Three of the 5 patients underwent previous autologous stem cell transplant, and 2 had a prior allogeneic stem cell transplant. There was a clear shift of PD1+ cells within the CD4+ T cell population in the HL patients compared to normal controls. PD-1 was significantly elevated in the peripheral blood CD4+ cells of HL patients compared to normal volunteers (mean MFI 1034 vs 123, p 〈 0.025). PD-1 was also elevated on the CD8+ T cells of the HL patients compared to normal volunteers (MFI 808 vs 221), but did not reach significance. There was no correlation between the level of PD-1 elevation on CD4+ or CD8+ cells, and prior autologous or allogeneic transplantation. Conclusion: PD-1 expression in peripheral blood CD4+ T cells may be a potential biomarker of systemic immune dysregulation in heavily pre-treated HL patients. PD-1 warrants further exploration in relapsed/refractory HL both as a potential biomarker, and as a target for directed immunotherapy. Additional studies by our group, investigating the role PD-1 as a biomarker in relapsed/refractory HL are ongoing. Disclosures: O'Connor: Spectrum: Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Consultancy, Research Funding.