ALBERT

Description: Abstract 4583 Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear. The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively. Leukemic cells from progressive (n=9) and non-progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. Dvl 1, Dvl 2, Dvl 3 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins. Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p

Description: Abstract 2927 The standard of care for patients (pts) with asymptomatic multiple myeloma (MM) stage I/II or a stable response after chemotherapy is “watchful waiting”, with conventional agents failing to demonstrate benefits as early or maintenance therapy. We report data from a randomized, open-label, phase II study of BLP25 liposome vaccine (L-BLP25, Stimuvax®), a therapeutic cancer vaccine (TCV) targeting the mucin 1 (MUC1) antigen, in pts with previously untreated, slowly progressive, asymptomatic MM I/II or MM II/III in stable response/plateau phase following anti-tumor therapy. Pts were randomized to L-BLP25 and either a single (Group [Gp] A) or multiple (Gp B) low dose(s) of cyclophosphamide (CP). In both Gps, L-BLP25 (930 μg) was administered weekly for 8 wk, followed by maintenance vaccination every 6 wk until disease progression requiring anti-tumor therapy. A single dose of CP (300 mg/m2, maximum total dose 600 mg) was given 3 days before the first L-BLP25 dose and, in Gp B, also prior to the vaccination at wk 5 and each maintenance phase vaccination. The primary objective was to investigate the immune response to L-BLP25. A specific induced immune response to MUC1 was defined as ≥2-fold increase over baseline and no peptide control in an IFN- γ ELISpot, lymphoproliferation or intracellular IFN- γ FACS assay following short-term in vitro stimulation of PBMCs with MUC1-derived peptides, on ≥2 assessments. Secondary objectives included safety/tolerability, quality of the immune response, linkage of immune response to HLA restriction, clinical effects, time to progression and time to anti-tumor therapy. Median data are presented. Thirty-four pts (age: 64 years; 15 male) were randomized and received treatment (Safety Analysis [SA] set; A/B = 17/17) of which 32 (17/15) pts met the pre-specified criteria for immunologic analysis (Immunological Diagnostic Analysis [IDA] set). The duration of MM (SA set) was 34 vs 37 months (Gp A vs B) at study entry. The proportion of pts with untreated stage I/II vs previously treated stage II/III MM was 29 vs 71% (Gp A) and 47 vs 53% (Gp B). Of the previously treated pts, 83% (Gp A) and 100% (Gp B) had received high-dose chemotherapy with autologous stem cell transplantation. In this analysis, all pts had reached ≥50 wk or had discontinued study treatment. Treatment duration was 54 vs 87 wk (Gp A vs B), with 15 vs 21 L-BLP25 vaccinations and 1 vs 11 CP infusions (SA set). Cumulative CP dose (first 50 wk) was 297 vs 1769 mg/m2 (Gp A vs B), corresponding to a relative dose intensity of 100 and 97%. As in previous studies, spontaneously induced specific MUC1 immune responses were seen frequently pre-vaccination (baseline Gp A/B = 59/47%). Specific induction/augmentation of the MUC1 response was seen in 53% of pts following L-BLP25 treatment with no difference between Gps A and B. Rates of induction/augmentation following vaccination were similar for pts with vs without a spontaneous (pre-vaccination) immune response. First immune responses occurred early (≤9 wk) in the course of treatment. Assessments of cytokines will be presented. Objective clinical responses (Bladé criteria) were not seen, and were not anticipated given the aim of TCVs is to stabilize disease rather than induce a response according to established chemotherapy criteria. In a preliminary analysis, reduction in the slope of on-study M-protein concentration over time compared with pre-study data was seen in 13 of 30 pts (10/13 previously untreated and 3/17 previously treated; updated analysis will be presented). There was no association between presence of a treatment-induced MUC1 response and on-study changes in M-protein (area under the curve at wk 26 [AUC26]; IDA set). However, on-study M-protein AUC26 was significantly lower in pts without vs with a spontaneous pre-vaccination MUC1 response (pooled data for both groups: AUC26 = -5.2 [n=13] vs 16.2 [n=16], p=0.015). However, these data should be interpreted with caution given the small number of pts. Treatment was generally well tolerated. One possibly treatment-related fatal event of encephalitis occurred in Gp B. In summary, L-BLP25 led to MUC1-specific immune responses in a large proportion of MM pts and was associated with emerging clinical effects on M-protein concentration over time. Our data suggest that pts with previously untreated, early-stage disease may be the most likely to benefit from L-BLP25 vaccination. Further investigation of L-BLP25 in MM is warranted. Disclosures: Off Label Use: The abstract reports the results of a Phase II investigative study of L-BLP25 in multiple myeloma. L-BLP25 is a therapeutic cancer vaccine targeting mucin 1, which is widely expressed on common cancers. Österborg:Merck GmbH: Research Funding. Forssmann:Merck Serono S.A., 9 Chemin des Mines, CH-1202 Geneva, Switzerland: Employment. Senger:Merck KGaA, Darmstadt, Germany: Employment. Schröder:Merck KGaA, Darmstadt, Germany: Employment. Mellstedt:Merck-Serono: Honoraria, Research Funding.

Description: Alemtuzumab is a humanized anti-CD52 antibody licensed for refractory B-cell chronic lymphocytic leukemia (B-CLL), when given intravenously at 30 mg thrice weekly. However, the intravenous route is associated with infusion-related reactions and is inconvenient. We measured blood concentrations in 30 relapsed patients treated with intravenous alemtuzumab and in 20 patients from a previously untreated group who received similar doses subcutaneously. Highest trough samples in the intravenous group were less than 0.5 μg/mL to 18.3 μg/mL (mean 5.4 μg/mL). The cumulative dose required to reach 1.0 μg/mL was 13 mg to 316 mg (mean 90 mg). Higher blood concentrations correlated with the achievement of better clinical responses and minimal residual disease. The highest measured concentrations in the subcutaneous group were similar (0.6 μg/mL to 24.8 μg/mL, mean 5.4 μg/mL). However, the cumulative dose to reach 1.0 μg/mL was higher: 146 mg to 1106 mg (mean 551 mg). No antiglobulin responses were detected in 30 patients given intravenous alemtuzumab whereas 2 of 32 patients given subcutaneous alemtuzumab made substantial anti-idiotype responses. Thus, subcutaneous alemtuzumab achieved concentrations similar to those for intravenous alemtuzumab, although with slightly higher cumulative doses. Subcutaneous alemtuzumab is more convenient and better tolerated but may be associated with some patients forming anti–alemtuzumab antibodies, particularly those patients who were previously untreated.

Description: Background: Multiple T cell abnormalities in Chronic Lymphocytic Leukemia (CLL) patients are well established. Compared to healthy individuals, total T lymphocyte count, CD8+ cells and CD4+ cells in CLL patients are increased and follow disease activity, i.e. increase at CLL progression. Major alterations in T cell functions and TCR repertoire have also been reported as well as T cells specifically recognizing the leukemic CLL cells. It may be hypothesized that aberrantly functioning T cells contribute to a microenvironment which may allow CLL cells to avoid apoptosis and/or support the proliferative capacity. However, the underlying mechanisms are largely unknown. Materials and Methods: T cells and CLL cells were highly purified from PBMC of 20 patients with indolent CLL as well as normal blood B- and T-cells from healthy control donors (n=7). Autologous T cells and B cells were co-cultured at different T:B ratios for 72 h in ordinary and transwell plates. Apoptosis of B cells were assayed using Annexin-V and supernatants were analysed by Luminex for the following cytokines: RANTES, IL-10, IL-8, TNF-α, IFN-γ, IL-4, MCP-1, CD40L and GM-CSF. Results: Apoptosis of CLL cells was significantly (p

Description: We have previously established the suitability of dendritic cells that have endocytosed apoptotic B-CLL cells (Apo-DC) as an antigen presentation platform to stimulate autologous antileukemic immune responses. The safety as well as immunological and clinical efficacy of Apo-DC vaccination was tested in a phase I/II clinical trial. CLL patients (Rai stage 0–2) who did not require concurrent therapy were leukapheresed and CD14+ monocytes as well as CD19+ B-CLL cells were purified immunomagnetically. Dendritic cells were generated ex vivo with GM-CSF and IL-4. Apoptotic bodies were obtained by irradiating B-CLL cells and overnight culture. DC were allowed to endocytose the apoptotic bodies and further matured with TNFα. The first cohort of 5 patients received 4 doses of the vaccine 2 weeks apart and a 5th dose at week 14. Each vaccine dose comprised of 107 autologous Apo-DC. The second cohort of 5 patients received the vaccine in a similar schedule with the addition of 75 μg of GM-CSF (Leukine™, Berlex Laboratories, Richmond VA) with every vaccine administration. Patients are scheduled to be monitored for a minimum of 52 weeks which has been completed for all of the patients in cohort 1 and is ongoing for cohort 2 The vaccine was well tolerated and occasional, low-grade toxicity was associated with the administration of GM-CSF. 4/5 patients in cohort 1 and 3/5 patients in cohort 2 responded immunologically in terms of increase in T cell proliferation or γ-IFN ELISPOT against autologous leukemic targets compared to prevaccination levels. Analysis of T cell responses at week 8 and week 16 following vaccination did not demonstrate an obvious augmentation of immunity by GM-CSF. One patient in cohort 1 demonstrated a transient decrease in WBC counts but no significant clinical responses have been noted thus far. Our results demonstrate that it is feasible to enrich monocyte precursors and generate Apo-DC for vaccinating patients with high count B-CLL. No significant toxicity was associated with this approach to vaccination therapy. The clinical efficacy of Apo-DC vaccination in CLL remains to be determined at the termination of the clinical trial.

Description: Background CLL patients (pts) have impaired humoral and cellular immune functions, which is largely due to profound defects of T-cells. Regulation and activation of T lymphocytes depend not only on T cell receptor signaling but also on co-signaling receptors delivering either inhibitory or stimulatory signals, known as immune checkpoints. CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) is transiently expressed on activated T cells, binding the same ligands as CD28, inhibiting T-cell activation. Similarly, programmed cell death protein 1 (PD-1) is expressed on activated CD4+ and CD8+ T cells inhibiting T-cell functions upon binding to the ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). CD137 is an inducible costimulatory receptor expressed by activated T cells. Dysregulated expression of immune checkpoint receptors on T cells of CLL pts may have an impact on T-cell responsiveness and might be a mechanism for the immune deficiency in the disease. Aim To evaluate the expression of the immune checkpoint molecules CTLA-4, PD-1 and CD137 as well as of the cell proliferation marker Ki67, the activation marker CD69 and of CD103, a marker expressed on regulatory T cells, in T cells from CLL pts in different disease phases. Methods Peripheral blood samples were obtained from 69 CLL pts and 13 healthy control donors (HD). Pts were sub-grouped according to disease phase: indolent vs progressive (i.e. fulfilling criteria for active disease). The expression of CTLA-4, PD-1, PD-L1, CD69, CD103, CD137 and Ki-67 was assessed by flow-cytometry on CD4+ and CD8+ T cells. We also analysed the change in expression of these markers on T cells after 72 hours of PHA stimulation. Results CLL pts (n=17) had a significanty higher percentage of proliferating (Ki67+) CD3+ cells compared to HD (n=7) (median 3.7% in progressive vs 1.7% in indolent CLL vs 0.9% in HD, p=0.004 and p=0.04, respectively) (Fig.1). Progressive CLL pts had a significantly higher percentage Ki67+ CD4+ compared to indolent pts as well as HD (p=0.007 and p=0.001, respectively). Both indolent and progressive pts had higher percentage of Ki67+ CD8+ T cells compared to HD (p=0.01 and p=0.03, respectively). The percentage of CTLA-4+ CD4+ and CTLA-4+ CD8+ cells was low in CLL pts as well as in HD. However, the percentage of PD-1+ CD4+ T cells was significantly higher in progressive (n=32) as compared to indolent (n=35) CLL pts (median 40.3% vs 23.3%, p

Description: Abstract 3603 Background: The receptor tyrosine kinase Ror1 is a tumor-associated molecule over-expressed in chronic lymphocytic leukemia (CLL) and a variety of other malignancies with potential implication for targeted immunotherapy. Little is known about the functional role of Ror1 in the leucomogenesis of CLL. Aims: To assess spontaneous T cell response against Ror1 in CLL patients. Methods: Autologous T cell response against Ror1 was studied in 9 CLL patients and 6 healthy subjects expressing the HLA-A2 allele. Four synthetic 9-mer HLA-A2 restricted peptides selected from different parts of the Ror1 molecule were loaded on dendritic cells and co-cultured with enriched autologous T cells. T cell response was determined by enumeration of the frequency of IFN-γ, IL-5 and IL-17A secreting T cells by ELISPOT. Cell proliferation was determined by 3H-thymidine incorporation. Results: Our results demonstrated a significantly higher frequencies of IFN-γ producing T cells (p