ALBERT

Description: Abstract 3727 Poster Board III-663 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Entinostat (SNDX-275) is a novel and potent DAC inhibitor that is selective for class I DACs and is currently undergoing pre-clinical and clinical testing in Hodgkin lymphoma (HL). Potent synergistic anti-tumor activity has been observed by combining less potent DAC inhibitors with bortezomib in pre-clinical models. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphoma, we evaluated the effects of Eentinostat as a single agent and in combination with bortezomib against B-cell non-Hodgkin's lymphoma (NHL) cell lines and primary NHL cells. Studies were conducted in a panel of 12 NHL cell lines representing various subtypes of B-cell lymphoma (i.e. DLBCL/ABC, DLBCL/GCB, Burkitt's, transformed and MCL), which included: rituximab-[chemotherapy]-sensitive cell lines (RSCL, Raji, RL and DHL-4), rituximab-[chemotherapy]-resistant cell lines (RRCL, Raji-4RH, Raji-2R, RL-4RH, and DHL-4 4RH), and primary lymphoma cells isolated from patients with various subtypes of NHL and HL. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells and patient-derived primary cells were exposed to entinostat at different doses (0.01 to 100uM) either alone or in combination with CDDP (1 to 100μM), doxorubicin (4 to 16μM), vincristine (1 to 5μM), or bortezomib (1 to 10nM). Anti-tumor activity was measured after a 24 or 48 hr incubation. In cell lines, changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. For patient-derived primary NHL cells, changes in ATP content (apoptosis) was determined using the cell titer glow assay. Entinostat was highly active in all the cell lines tested including rituximab-[chemotherapy]-resistant cell lines. The IC50 of Entinostat in the majority of the cells tested was 0.5 to 5uM at 48 hrs. Similar findings were observed in primary tumor cells derived from lymphoma patients. In addition, synergistic activity was observed by combining entinostat and bortezomib in both NHL cell lines, as well as in primary NHL/HL tumor specimens. A lesser degree of augmented anti-tumor activity was also observed when entinostat was combined with cisplatin or doxorubicin (but not vincristine). In summary, our data suggests that entinostat is a novel and potent DAC inhibitor with a wide therapeutic spectrum. Entinostat is capable of inducing cell death against various subtypes of B-cell lymphoma cell lines including RSCL, RRCL, as well as patient-derived primary tumor cells and augments the anti-tumor effects of bortezomib and other chemotherapeutic agents. Given the isoform selectivity of entinostat, the results indicate that HDAC1 and 2 may be the key targets of DAC inhibitors in HL and NHL cells. Ongoing studies are evaluating the mechanisms responsible for the synergistic effects of entinostat plus chemotherapy and will be updated at the annual meeting. Current findings strongly suggest that entinostat added to bortezomib and/or other chemo agents may become a novel and potent strategy in the treatment of aggressive and indolent NHL and HL in the future. Disclosures: No relevant conflicts of interest to declare.

Description: Cancer cells, including B-cell lymphoproliferative disorders, sculpt their phenotype in an attempt to escape not only immune-surveillance but also evade the anti-tumor activity of biological agents and/or chemotherapy drugs including rituximab. In an attempt to study the mechanisms that regulate the emergence of rituximab resistance, we developed several rituximab-resistant cell lines and demonstrated that the emergence of a rituximab-resistant phenotype was associated with global down regulation of CD20 antigen and unexpectedly an increase in CD52 antigen. Validation of our findings in more clinically relevant settings is important to support alemtuzumab-based clinical studies in rituximab-resistant B-cell malignancies. To this end, we retrospectively studied changes in CD20 expression over time in alemtuzumab treated-patients with rituximab-fludarabine (RF) refractory CLL. Patients were identified using the institute tumor registry and pharmacy electronic database. Demographic characteristics, treatment history, outcome data were obtained for each patient. In addition, CD20 expression in CLL cells obtained from flow cytometry analysis performed on peripheral blood, bone marrow and tissue was reviewed. A total of 16 patients with RF refractory CLL treated with alemtuzumab were included in the analysis, 13 males and 3 females, the median age at the time of diagnosis was 55.5 yrs +/− 10.47stv, and most of the patients had a good PS (0) at the time of treatment with alemtuzumab (81%). The response rate to alemtuzumab was 56.3%, with 7 (43.5%) patients achieving a complete response (CR). After a median follow up period of 88.5 months, 7 patients are still alive, 3 are free of disease. Complete flow cytometry data was available only for 9 patients. A down regulation of CD20 antigen expression was observed among 5/9 patients in blood, bone marrow and/or tissue over time when compared to baseline CD20 levels. Following alemtuzumab therapy, CR was achieved in 3/5 patients with CD20 down-regulation versus 1/4 patients with steady levels of CD20. Alemtuzumab-treated CLL patients with CD20 down-regulation had a longer overall survival (141 months) than alemtuzumab treated CLL patients with steady levels of CD20 antigen overtime (116 months, Log Rank P = 0.026). Our data suggest, that emergence of rituximab-fludarabine resistance is associated with changes in the expression of CD20 antigen. A response to alemtuzumab in this setting appears to be higher than historical controls (2% CR). Refractory CLL patients with decreased levels of surface CD20 appears to respond better to alemtuzumab and have a longer overall survival than patients with steady CD20 levels. Similarly to what we have observed in our pre-clinical models, an increase in CD52 expression in rituximab-fludarabine refractory patients with CD20 down regulation could explain the higher response rate observed in this small group of highly selected patients. Detailed monitoring of CD20 and CD52 levels in CLL patients before, during and after treatment with rituximab-based regimens is warranted in an attempt to identify patients that can benefit from almetuzumab-based therapies.

Description: Despite the improved outcome in patients with DLBCL treated with rituximab (R) in combination with systemic chemotherapy (R + chemotherapy), a significant number of patients either relapse or fail to respond as a consequence of resistant disease. HDC and ASCT is the best therapeutic strategy to rescue relapsed/refractory DLBCL. It has been postulated that R+chemotherapy may lead to the selection of highly resistant lymphoma cells diminishing the clinical benefit of HDC and ASCT. Preliminary data from the CORAL study (Gisselbrecht et al Blood2007; 11:517a) suggest that overall response rates (ORR) and 2-year event free survival (EFS) are lower in R+chemotherapy relapsed/ refractory DLBCL when compared to DLBCL treated with chemotherapy alone. However the second randomization of this study to observation versus R-maintenance may affect the interpretation of the data. We retrospectively studied the difference in the outcomes of relapsed/refractory DLBCL patients following HDC and ASCT according to the front line therapy utilized (R+chemotherapy versus chemotherapy). Using the Roswell Park Cancer Institute (RPCI) Tumor Registry and the RPCI Blood and Marrow Transplant (BMT) Database we identified 130 patients with relapsed/refractory NHL who underwent for HDC + ASCT from 1991 to 2008. After excluding patients with a diagnosis other than B-cell DLBCL (patients with transformed NHL were excluded) and those patients receiving allo-BMT after progression from ASCT, the analysis included 63 refractory/ relapsed DLBCL. Demographic characteristics, clinical data, treatment history in the front line and salvage setting were collected. In addition response to salvage therapy and disease status at day +100 from ASCT was recorded for each subject. Progression free and overall survival were calculated from ASCT. Differences in clinical outcomes between patients receiving R as part of first line or salvage treatment and those treated with chemotherapy alone were evaluated by multivariate analysis, adjusting for significant univariate predictors of survival. The patient cohort included 34 males and 29 females with median age of 46 yrs (14.4 to 69.4). Two-thirds of the patients had advance disease and the majority had a Karnofsky performance status (KPS) of 80–100% at diagnosis. R+chemotherapy was given in the front line setting to 25 pts and while 38 received chemotherapy alone. In the salvage setting, 35 pts (55%) received R+chemotherapy. Most relapses (44 pts) occurred within 6 months of completion of front line therapy (17 pts with vs. 27 pts without R). The use of R in the front line setting was associated with significantly higher response rates (PR + CR) to salvage chemotherapy (P = 0.036) and better disease control on day +100 post-ASCT (P = 0.016) when compared to chemotherapy alone. In our cohort, there have been 32 deaths, 23 in chemotherapy treated DLBCL in contrast to 9 deaths in R+chemotherapy treated patients There was a significantly higher response rate post-ASCT for R+chemotherapy treated (as front-line or salvage) DLBCL versus chemotherapy alone (P = 0.007). A multivariate analysis demonstrated that achieving a CR pre-ASCT was the most important predictor of post-ASCT progression free and overall survival . In summary, our data suggest that the use of R + chemotherapy during frontline therapy and in the salvage setting yields better disease control and less incidence of chemo-resistant disease at the time of BMT. Applying the natural selection theory, the use of R+chemotherapy is expected to result in the development of resistant lymphomas. The length of time and the amount of R therapy that will render lymphoma cells resistant to chemo-immunotherapy remain to be determined. Standard doses of R (6 to 8 doses) do not appear to affect response to salvage therapy or autologous BMT outcomes. In our single institution analysis over the last 18 years, it appears that HDC + ASCT is an effective and viable option for patients with R +/− chemotherapy relapsed/refractory DLBCL.