ALBERT

Description: Obatoclax antagonizes the BH3-binding groove of the bcl-2 family of anti apoptotic proteins. It is active in chronic lymphocytic leukemia(CLL; O’Brien et al, ASH 2005) with a recommended phase II dose of 28 mg/m2 given over 3 h every 3 weeks with DLT of grade 3 infusional CNS toxicities. We evaluated prolonged infusions to minimize these toxicities while maintaining clinical activity. An initial trial established a Phase II dose of 28 mg/m2 over 24 h every 2 weeks with dose limiting toxicities (DLTs) at 40 mg/m2 and responses in 4/8 MDS patients (Borthakur et al., ASH 2006). We report on 21 patients who received obatoclax 20–28 mg/m2/24 h weekly (n=9) or 20 mg/m2/24 h for 2, 3 or 4 consecutive days every 2–3 weeks (n=12). The final dose level of 28 mg/m2/24 h for 4 days is now open. Median age was 61 (range 26–75) and 11 patients were male. Diagnoses included: acute myelogenous leukemia (AML; 13), myelodysplastic syndromes (MDS; 6), acute lymphocytic leukemia (ALL; 1) and CLL (1). A total of 180 infusions of 24 h were administered. The most common adverse events (AE) were euphoric mood (57%), fatigue (57%), febrile neutropenia (43%), gait disturbance (43%), chills (33%), diarrhea (33%), nausea (33%), somnolence (33%), dizziness (29%). All were of Grades 1–2 with the exception of 7 grade 3 AEs of febrile neutropenia (unrelated to obatoclax administration) and 1 each of diarrhea, fatigue and gait disturbance. There were no DLTs. Multiple day infusions were well tolerated; for the 20 mg/m2/24 h x 4days dose level, 4/6 patients received ≥ 4 cycles, with two ongoing at this time. Plasma concentrations of obatoclax reached a steady state before end of infusion. Mean Cmax values at 20 mg/m2/24 h during1x24, 2x24, 3x24,4x24 h, and 28 mg/m2/24 h during 1x24 h infusions was 15.3, 8.4, 10.1, 15.7 and 10.8 ng/mL, respectively. The mean AUC(0–tlast) values at 20 mg/m2/24 h associated with 1x24, 2x24, 3x24, 4x24 h, and 28 mg/m2/24 h associated with 1x24 h infusions were 264.3, 396.9, 624.3, 1109.3 and 211.1 ng · hr/mL, respectively, increasing as expected with the duration of the infusion. One patient with treatment-related AML with a t(9;11)(p22;q23) translocation achieved a cytogenetic complete response (CR) with complete hematological recovery and transfusion independence on Day 9 following the start of weekly 24 h infusions of obatoclax. This CR was sustained 〉 8 months while the patient received a total of 35 weekly infusions of 20 mg/m2 without cumulative toxicities. Conclusions: obatoclax can be administered by prolonged infusions without eliciting novel or cumulative toxicities. The dramatic response observed in a patient with AML with immediate recovery of peripheral blood counts supports that the cytotoxic effects of obatoclax are specific to malignant cells while sparing normal bone marrow, as did our earlier reports of improvement in cytopenias in patients with MDS and CLL. The infusional schedules described here will be attractive to combine with standard chemotherapy regimens for AML and other indications to improve treatment outcomes where they may be limited by overexpression of Bcl-2 family members.

Description: The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Description: Background .Chronic GVHD (cGVHD) is the most common and severe late complication after allogeneic stem cell transplantation (SCT), because of increasing use of matched unrelated donors, peripheral blood stem cells and reduced intensity conditioning (RIC). Impaired immune reconstitution and unbalanced Th1-type or Th2-type immune response with cytokines dysregulation seem to play a key role in the pathogenesis of chronic GVHD (cGVHD). Aims. In order to study the immunologic mechanisms involved in cGVHD, we prospectively evaluated the Th1 (TNF-alpha, IFN-gamma) and Th2 (IL-4, IL-6, IL-10) cytokine patterns and lymphocyte subsets in the setting of allogeneic SCT-RIC. Patients and methods. We assessed by ELISA the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, IL-10 and soluble tumor necrosis factor receptors I and II (sTNF-R) in 8 healthy donors and in 19 patients undergoing allogeneic SCT-RIC (13 patients with related and 6 patients with unrelated donor). Serum levels were assessed before transplantation and monthly from second to twelfth month after SCT. Total lymphocytes and their subsets with different co-stimulatory molecules (CD4, CD8, CD19, CD28/3,CD25/4, CD134/4, CD152/3, CD16/56, CD4/45ro, CD4/45ra, CD8/45ro, CD8/45ra) were evaluated by flow cytometry in peripheral blood (PB) monthly after SCT. Cyclosporine A was used as GVHD prophylaxis in all patients from third month until its tapering or until the beginning of the therapy against extensive cGVHD. Wilcoxon test was used for statistical analysis of the data. Results. Twelve out of 19 patients developed cGVHD at a median time of 7 months (range, 6–10); cGVHD was extensive in 8 patients at a median time of 8 months (range, 6–12). The levels of cytokines did not differ significantly in patients pre-SCT and healthy donors. Patients with cGVHD differed from those without cGVHD because of: significantly higher levels of TNF-alpha from third to sixth month after SCT (3rd, p=0.003; 4th p=0.001; 5th, p=0.0005; 6th, p=0.01); significantly higher levels of sTNF-R II at 6th (p=0.01); significantly higher levels of IL-10 (p= 0.004) at 4th month; significantly lower number of NK cells in PB from third to sixth month after SCT (3rd, p=0.004; 4th p=0.009; 5th, p=0.0007; 6th, p=0.0003); significantly lower number of CD 152/3 cells in PB from third to sixth month after SCT (3rd, p=0.009; 4th p=0.0004; 5th, p=0.0004; 6th, p=0.007); significantly lower number of CD 4/25 cells in PB at 4th (p=0.0004) and 5th (p=0.01) Discussion. Our sequential study showed: increased levels of Th1 (TNF-alpha) and Th2 (IL-10) cytokines with different kinetics after SCT and before the onset of cGVHD; a decrease of NK and T cells with regulatory molecules such as CD152 after SCT and before the onset of cGVHD. These results suggest an overall prevalence of a TNF-alpha oriented response in patients with cGVHD. Defects of immunoregulatory cells could be related to these fluctuating and unbalanced cytokine patterns. However, further studies with more patients are required to support these preliminary results.

Description: Although CEBPA mutations (CEBPA+) have been reported to predict favorable outcome in CN-AML, their prognostic value has not been evaluated in the context of such established prognostic molecular markers in CN-AML as the combination of FLT3-ITD and NPM1 mutational status and BAALC and ERG expression. 169 adults aged

Description: Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4+ T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

Description: Background: Factors influencing rates of alloimmunization to antigens on transfused red blood cells (RBC alloimmunization) are poorly defined. In particular, the role of the spleen with respect to alloantibody formation is unclear, with conflicting clinical reports in the literature. Moreover, the complexities of multiply mismatched antigens and antigen priming due to transfusions prior to splenectomy make human studies difficult to interpret. To better define the role of the spleen in RBC alloimmunization, we utilized a murine model of transfusion medicine (with the model RBC antigen mHEL (membrane bound hen egg lysozyme)). Methods: Cohorts of splenectomized and non-splenectomized mice (C57BL/6 × B10.BR) were transfused with the human dose equivalent of 1 unit of leukoreduced mHEL RBCs. RBC alloimmunization was assessed by anti-HEL IgG specific ELISA. The role of antigen-specific CD4+ T cells was studied by the adoptive transfer of 1.5 × 106 HEL-specific CFSE-labeled CD4+ T cells from 3A9 TCR transgenic donors. Adoptively transferred cells were visualized using a congenic marker (Thy1.1); enumeration and division of these cells were monitored by flow cytometry in liver, spleen (if applicable), and lymph node preparations. Results: Splenectomy dramatically decreased RBC alloimmunization; 14 of 14 splenectomized mice (from 3 experiments) had undetectable to very low levels of anti-HEL IgG following transfusion with mHEL RBCs (average 6.3 fold less than non-splenectomized mice, 95% C.I. 4.6). Moreover, ten of ten splenectomized mice failed to make detectable levels of anti-HEL IgG even following the adoptive transfer of HEL-specific CD4+ T cells. In comparison, elevating the precursor frequency of HEL-specific CD4+ T cells increased RBC alloimmunization by 10,000 fold in non-splenectomized mice. Proliferation and division of CD4+ T cells were detectable in both spleen and liver preparations of non-splenectomized mice by day 3 following transfusion; in contrast, no expansion nor division of CD4+ T cells was seen in liver nor lymphatic preparations of splenectomized animals. Conclusions: The low level of RBC alloimmunization seen in non-splenectomized mice in this system is limited by existing CD4+ helper T cell responses, as increasing the naive helper T cell precursor frequency dramatically increased RBC alloimmunization. Furthermore, the spleen itself is critical to CD4+ helper T cell function during alloimmunization given that, in splenectomized mice, adoptively transferred HEL-specific CD4+ T cells fail to expand, divide, or stimulate production of detectable alloantibody. Ongoing studies are investigating the phenotype of HEL-specific CD4+ T cells (as effector cells, anergic cells, or regulatory cells) in splenectomized and non-splenectomized mice. These studies have implications for preventing alloimmunization in transfusion-dependent patient populations.

Description: Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

Description: FL, the most prevalent indolent lymphoma in the US, is less common in non-White populations including African Americans (AA) and Hispanics (H), limiting our knowledge about its natural history in these patients (pts). In addition, since minority pts are often under-represented in clinical trials, it is unclear whether our current treatment paradigm in FL applies to them. The NLCS is a prospective, longitudinal, multi-center, observational study that enrolls pts with newly diagnosed FL and collects data on their disease presentation, treatment patterns, and outcomes. Using NLCS, we evaluated demographics, disease presentations and patterns of care in FL amongst AA, H, and white (W) pts. As of 1/31/07, 87 AA, 118 H, and 2314 W pts were enrolled in the NLCS. AA accounted for 3.4% and H for 4.6% of total registered pts. Despite the small numbers, this represents the largest US-based database available for AA and H pts with FL. Data collection included information on grade, stage, B symptoms, FLIPI, and treatment choice. Either Chi-square or Fisher’s exact test was used to assess the correlations depending on the sample size of the test. Below are the demographics and disease characterictics of analyzed pts: WhiteN=2314 (%) African-AmericanN=87 (%) HispanicN=118 (%) Age (yr) 45 216 ( 9.3) 23 (26.4) 24 (20.3) 45–59 774 (33.5) 30 (34.5) 39 (33.1) 60–74 856 (37.0) 22 (25.3) 41 (34.8) 75+ 468 (20.2) 12 (13.8) 13 (11.9) Sex Male 1138 (49.2) 37 (42.5) 52 (44.1) Stage I 419 (18.1) 12 (13.8) 21 (18.0) II 351 (15.2) 12 (13.8) 18 (15.4) III 683 (29.6) 25 (28.7) 39 (33.3) IV 840 (36.7) 38 (43.7) 37 (31.6) Grade 1 976 (42.3) 46 (52.9) 37 (31.6) 2 643 (27.8) 19 (21.8) 35 (29.9) 3 428 (18.5) 10 (11.5) 31 (26.5) Unknown 216 ( 9.4) 11 (12.6) 11 ( 9.4) Mixed 47 ( 2.0) 1 ( 1.2) 3 ( 2.6) FLIPI Good 709 (36.1) 22 (28.6) 34 (37.8) Intermediate 595 (30.3) 17 (22.1) 29 (32.2) Poor 659 (33.6) 38 (49.4) 27 (30.0) B-symptoms Yes 584 (25.3) 27 (31.0) 34 (29.1) Two statistically significant differences in baseline characteristics were that AA pts were younger and had a poorer FLIPI at diagnosis compared to W pts. Looking at initial treatment patterns, the only difference was the use of anthracyclines among those receiving chemotherapy, with W pts more likely to receive an anthracycline-based regimen than AA pts (65.8% vs. 46.8%, p=0.01). The higher use of anthracyclines in W compared to AA pts persisted regardless of FLIPI score. Aside from anthracycline use, there were no major differences in treatment approaches and choices according to grade of disease between AA, H, or W pts. AA pts with grades 1 or 2 FL received less anthracyclines compared to W pts (37.5% vs. 56.4%, p=0.04). The small number of AA pts with grade 3 disease precluded a meaningful comparison with W pts in this subgroup. In conclusion, while the numbers are small, the NLCS provides the largest prospective registry information on AA and H pts. AA pts, who were younger and had a poorer FLIPI at diagnosis, also received less anthracycline-based therapies than W pts, whereas H and W pts were treated similarly. Further analyses are required to understand the underlying reasons for these observed differences. Long-term follow-up will determine if outcomes vary in these pt cohorts.

Description: BACKGROUND: The role of rituximab (R) as a component of therapy for relapsed and refractory diffuse large B cell lymphoma (DLBCL) is controversial. We have previously reported that the addition of rituximab to ICE (RICE) second-line chemotherapy improved outcomes (Kewalramani, 2004). Furthermore, a small phase II study performed at Stanford University (Horwitz et al, 2004) suggested improvement in event free survival (EFS) and overall survival (OS) with the addition of rituximab maintenance therapy following high dose therapy and autologous stem cell rescue (HDT/ASCR) in rituximab naïve patients. However, it is unknown if there is benefit of rituximab maintenace in patients (pts) receiving rituximab as part of pre-HDT cytoreduction. METHODS: We retrospectively identified pts with relapsed and refractory DLBCL who underwent uniform pre-HDT/ASCR cytoreduction with RICE between 1999 and 2006. Pts proceeded to HDT/ASCR if they had a response to RICE chemotherapy. The earlier pts (n=38) did not receive any maintenance (NM) and a subsequent cohort (n=43) was treated with post ASCR rituximab (MR) on one of two regimens (R wkly x 4 at day + 42 and day + 180, n=26 or R q8 wks x 6 starting on day+29, n=17). RESULTS: Median follow up for surviving pts is 2.9 yrs: 4 yrs in the NM group; and 2.2 yrs in the MR group. The 3 yrs EFS is 43% and 84% and OS is 59% and 89% for the NM and MR groups, respectively. A Cox regression model was constructed to include disease status (relapsed v refractory), maintenance rituximab (yes v no) and second-line IPI (LR v LI-HR). Disease status (p = .04) and rituximab maintenance (p = .003) were significant for OS. Only maintenance rituximab was significant for EFS (p 〈 .001) (Figure 1). In analyzing patients who did not receive maintenance therapy by second-line IPI low risk (0–1) and low-intermediate risk-high risk (2–5), as expected, there is a survival difference which approaches significance (p = 0.074) in favor of the low risk group. Maintenance rituximab eliminated the adverse impact of the second-line IPI on EFS and OS for pts with LI to HR disease. For pts with LR disease maintenance rituximab did not influence outcome. It has been previously reported that patients who receive post-transplant rituximab maintenance experience high rates of neutropenia. We noted 16 patients who experienced asymptomatic afebrile neutropenia (Grade III/IV). One pt (2.3%) required hospital readmission post transplant related to fever and neutropenia. Neutropenia was noted early in the treatment course of both regimens. Growth factors were used at the discretion of the physician. CONCLUSION: Maintenance rituximab therapy post HDT/ASCR was well tolerated by patients and in this small sample significantly associated with improvement in EFS and OS. A prospective trial is currently underway to fully evaluate maintenance therapy versus observation (CORAL 50-03B). Figure Figure Patient characteristics RICE Alone RICE + Maintenance Total 38 43 Male 23 22 Female 15 21 Median Age (range) 52 (26–73) 48 (22–69) Disease Status Relapsed 27 18 Refractory 11 25 IPI LR 11 24 LIR-HR 27 19