ALBERT

Description: Purpose: To estimate the maximum tolerated duration (MTD) of gemcitabine given as a continuous infusion in combination with fludarabine and mitoxantrone for patients with relapsed or refractory leukemia. Patients and Methods: Patients received gemcitabine at 10mg/m2/min (day 1) with fludarabine at 25mg/m2/day for 5 days (days 1–5) and mitoxantrone at 10mg/m2/day for 3 days (days 1–3). The duration of gemcitabine infusion was escalated using the modified continuous assessment method (mCRM) at 3 hour increments (3, 6, 9, 12, and 15 hours). This three-drug regimen was build upon our previous studies with fludarabine, mitoxantrone and continuous infusion gemcitabine (Blood2003;102:250b Abstract). The MTD was estimated as the duration of gemcitabine infusion in combination with fludarabine and mitoxantrone at which 33% of the patients would experience a dose limiting toxicity (DLT). DLT was defined as either neutropenia for more than 28 days with less than 5% blasts in the marrow, a non-hematologic grade 4 toxicity lasting ≥ 3 days, or a non-hematologic grade 3 toxicity lasting ≥ 7 days. Results: Ten patients with relapsed or refractory leukemia have been treated so far since Aug 2004 and fully evaluated. The median age was 55 years (range 30 – 67). The median number of prior regimens was 3 (range 1 – 5) and the median range of prior cycles was 4 (range 1 – 6). As expected, hematologic toxicity, mucositis and nausea were common but did not meet criteria for DLT in any patient. One patient had parainfluenza lung infection with alveolar hemorrhage and this was the only DLT on the study. 2/10 patients achieved CR. Both patients are alive, 20 weeks and 52 weeks later, respectively but have received further therapy in the form of chemotherapy or allogeneic stem cell transplantation. Toxicity by Duration of Gemcitabine Infusion Duration of Infusion (hrs) # of Patients with DLT/Total Probability of Toxicity at this Dose Probability that Dose is MTD 3 0/1 0.001 0.0 6 0/2 0.009 0.005 9 0/3 0.1 0.22 12 1/4 0.36 0.59 15 0/0 0.6 0.18 Conclusion: Only one patient on this study had a DLT. This combination therapy is myelotoxic and has some anti-leukemia activity with 2/10 patients achieving CR. The duration of gemcitabine infusion as part of the above three drug regimen will be studied further on this ongoing study to more accurately estimate the MTD and determine if phase II trials are warranted.

Description: Abstract 1626 Background: Diffuse Large B-cell Lymphoma (DLBCL) patients with primary refractory or multiply relapsed disease have limited treatment options. Therefore it is imperative that novel drugs be tested to find new, effective approaches for these patients. With evidence demonstrating that increased vascularity and pro-angiogenic factors correlate with adverse prognosis in the hematologic malignancies, angiogenesis has emerged as a novel therapeutic target. PTK787/ZK222584 is an orally active amino-phthalazin that inhibits all known tyrosine kinase receptors of vascular endothelial growth factor (VEGF). The efficacy, safety and tolerability of PTK787/ZK222584 in patients with rel/ref DLBCL was evaluated in this non-randomized phase II study. Patients and Methods: Patients with relapsed or refractory DLBCL were eligible. Additional key inclusion criteria were negative proteinuria, and normal renal and liver function. Central nervous system disease, prior allogeneic transplant, recent chemotherapy, or previous anti-VEGF therapy excluded subjects from enrollment. All patients inititated PTK787/ZK222584 at a dose of 750mg by mouth (PO) daily on day 1 of a 28 day cycle. Drug dose was increased weekly, initially to a dose of 1000mg PO daily and then to a target dose of 1250mg daily unless a grade ≥2 toxicity (using NCI CTC V3.0) developed. Primary endpoint was response (complete response (CR) + partial response (PR)); secondary endpoints included safety and tolerability. Results: Twenty patients (11 female) with a median age of 60 years were enrolled. Sixty three percent of patients had received 〉3 prior therapies and 26% had prior autologous stem cell transplantation. Patients received a median of 3 cycles of PTK787/ZK222584 (range 0–6). Ten patients were on study drug for at least 3 cycles and are evaluable for response: one patient had a CR, four patients had stable disease (SD), and the remaining 5 patients had progressive disease. The patient with a CR subsequently underwent autologous stem cell transplantation and was alive without evidence of disease 64 months after study completion. One patient with SD withdrew to pursue other treatment options and the other 3 continued study drug, but all developed progressive disease within the next 3 months. Of the 10 patients who withdrew prior to completing 3 cycles, six withdrew after a median of 1 cycle because of rapidly progressive disease. Three withdrew because of adverse events of sepsis resulting in death in one patient, altered mental status despite dose reductions in one, and recurrent grade 3 transaminitis in the third. One patient withdrew to pursue other treatment options. Eighteen of the 20 patients enrolled escalated to the full target dose of 1250mg, but 6 required at least 1 dose reduction due to adverse events (sepsis, vertigo, transaminitis, rash or fatigue). There were no episodes of grade 3 / 4 proteinuria but grade 3 / 4 hypertension occurred in 10% and grade 3 / 4 thrombocytopenia in 20%, with the remainder of grade 3 / 4 toxicities occurring in

Description: B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

Description: Antiplatelet antibody in the serum of a fatal case of post-transfusion purpura showed specificity for the PlA1 platelet isoantigen in that the antibody was adsorbed by all PlA1-positive platelets tested but not by PlA1-negative platelets. However, some PlA1-positive platelets that fixed complement with the reference anti-PlA1 did not fix complement with this patient’s antibody. When used with these platelets, the patient’s antibody competitively blocked the complement-fixing activity of reference anti-PlA1. There was no evidence for lack of antigenic sites on the noncomplement-fixing platelets or for antibodies with more than one specificity in the patient’s serum. These studies, therefore, indicated that the complement-fixing activity of different examples of anti-PlA1 is variable, and that this variation is probably due to distribution of PlA1 antigenic sites on cell surfaces rather than to differences in antigenic structure that affect antibody affinity.

Description: B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients’ samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.

Description: Background: Although most malignancies are associated with decreased numbers of circulating T cells, in CLL they are elevated 2 to 4 times normal. Rather than promoting an anti-tumor response, this increased population of T cells may contribute to a tumor microenvironment that fosters progression of the malignant clone. Immunocompetent individuals show a wide repertoire of antigen specificity in both CD4+ and CD8+ T cells, but the T cell repertoire is significantly restricted in CLL. This restriction of the T cell repertoire may be an important cause of infectious morbidity in patients with CLL. To better understand these T cell abnormalities, we enumerated T cell subsets and determined T cell receptor diversity in 18 untreated patients with CLL. Methods: T cell subsets were enumerated from peripheral blood using highly sensitive 6-color flow cytometry. The T cell repertoire was determined for 23 T cell receptor variable β chain families (TCRvβ) in purified CD4+ and CD8+ T cells. These T cell subsets were considered separately because differential restriction of the CD4+ and CD8+ subsets has been reported previously. A PCR-based spectratype assay was used to analyze the length distribution of the TCR complementarity-determining region 3 (CDR3). A limitation of prior reports using spectratype assays was that adequately complex statistical models did not exist to simultaneously analyze the highly diverse vβ families. We addressed this limitation by using a recently-developed statistical method for spectratype analysis (Bioinformatics. 21:3394–400). Briefly, for each vβ family, the divergence from an expected reference distribution was calculated. A divergence coefficient was determined for each vβ family, and the mean divergence of all 23 vβ families was calculated. This allowed for statistical comparisons among individual patients and specific vβ families. To our knowledge, we are the first group to apply this powerful methodology to the analysis of T cell repertoires in patients with CLL. Results: We found both the CD4+ and CD8+ subsets to be expanded (mean #/μL ± SD: 1134 ± 646 and 768 ± 716, respectively; reference normal CD4+ range 401–1532, CD8+ 152–838). The absolute number of CD4+ and CD8+ T cells was greater in patients with higher absolute CLL lymphocyte counts (p = 0.018, r2 = 0.30, and p = 0.23, r2 = 0.09, respectively, linear regression). The CD4:CD8 ratio was lower in IgVH unmutated subjects (mutated 2.6, umutated 1.7, p = 0.09, two-tailed t-test assuming unequal variances). Though prior reports have disagreed on whether CD4+ or CD8+ subsets show greater restriction of clonality, we observed striking clonal restriction of CD8+ but not CD4+ T cells (p 〈 1×10−7, 2 sided t-test assuming unequal variances). There was a trend toward greater restriction of the CD8+ subset among patients with IgVH unmutated and Zap70+ CLL, but there was no correlation with lymphocyte doubling time. Conclusions: In this cohort of 18 untreated patients with CLL, there was a greater proportional increase compared to reference standards of CD8+ versus CD4+ T cells. However, the increase in CD4+, but not CD8+, T cell numbers was significantly correlated to total CLL lymphocyte count. This observation suggests that expansion of the CD4+ T cell pool observed in CLL is proportional to leukemic burden. The restriction of TCRvβ was limited to CD8+ T cells and that this effect was independent of the size of the abnormal clone. Taken together, these findings suggest different mechanisms of dysregulation of CD4+ and CD8+ T cell subsets in CLL.

Description: Abstract 4935 Background: Myelodysplasticsyndromes (MDS) are a heterogeneous group of hematopoietic diseases characterized by peripheral blood cytopenias and increased apoptosis of precursor cells in the marrow. Current therapies are focused on disease control, with no cure other than allogeneic stem cell transplantation. Decitabine is known to act as a hypomethylator while lenalidomide has multiple affects, including actions on the stromal marrow cell interaction. It is hypothesized that the combination of decitabine with lenalidomide may show synergistic results. Thus we performed a phase 1 study to estimate the maximally tolerated dose of lenalidomide in combination with decitabine in patients with high risk MDS. Methods: Adults with primary or secondary MDS, including all subcategories and IPSS risk categories of INTERM-2 or High were eligible, including those who had prior therapy even if they progressed with prior exposure to hypomethylators. Patients also must have had CrCl 3 60ml/min, and liver function test 2 3 × ULN. Following a standard phase 1 3+3 design, patients received decitabine 20mg/m2 over one hour intravenously daily for 5 days, with cycles repeated every 28 days. Lenalidomide was given in cohorts at doses of 5mg, 10mg, or 15mg daily for days 1–21 of the 28 day cycle. Patients were analyzed during the first cycle for dose limiting toxicity and responders that did not have a DLT could continue until progression or toxicity required discontinuation. DLT was defined as any non-heme grade 3 toxicity (CTC v. 4) that was study related lasting 3 7 days or grade 4 of any duration or Neutropenia or thrombocytopenia 3 grade 3 due to study agents 〉14 days. Results: Patients and disease characteristics are noted in the table below. Twelve evaluable patients were treated, 5 had prior therapy (all included a hypomethylator). The combination seems tolerable, though toxicities included the following: 4 patients had a grade 3 febrile neutropenia (2 in cohorts 1 and 3 each), 2 with grade 2 rash (1 cohorts 1 and 3 each), and 4 with grade 3 anorexia/fatigue (1 cohort 1, 2 cohort 2, 1 cohort 3). With median duration of follow up for survivors of 6 months (2–38months) the median number of cycles of therapy was 4 (1–16) with 3 continuing on therapy at this time. Responses were noted at all dose levels, including those with prior hypomethylator exposure (see table 1): 3 (25%) complete responders, 1 partial responder (8%), and 3 (25%) with stable disease. The Median duration of response was 5 months (1–21) with 3 continuing in response. Conclusion: In patients with high risk MDS, the combination of decitabine and lenalidomide is tolerable and provides encouraging response rates in this population. The MTD was determined as decitabine 20mg/m2 daily for 5 days with lenalidomide 10mg daily for days 1–21 of a 28 day cycle. Larger studies of this combination are warranted. *MTD Disclosures: Rizzieri: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Beaven:Celgene: Research Funding.

Description: Abstract 4948 Background: MDS (Myelodysplastic syndrome) is a hematologic malignancy in which one or multiple conditions affects the fabrication (or dysplasia) of the myeloid class of blood cells. While treatable, therapies provide short term benefit and are not considered curable, short of an allogeneic stem cell transplant. The RAS pathway has been noted to be abnormally regulated in many malignancies, including MDS and AML. Thus, we performed a phase 2 efficacy study using sorafenib, a RAS inhibitor, for patients with MDS by WHO criterion. Methods: Adults with primary or secondary MDS, estimated CrCl 〉30 ml/min, and liver function tests 2 2. 5 × ULN were eligible. Patients of all IPSS risk categories were included. Patients received Sorafenib 400mg po bid daily for 28 days for a 28 day cycle (continuous therapy) and continued till progression or withdrawal for toxicity. Patients were evaluated after the first 2 cycles for response and then every 3 cycles thereafter until an endpoint was reached (up to five years). Results: Patient and disease characteristics are noted in the table below. Sixteen evaluable patients were treated on study: median 71 (range 56–88 years), median time from diagnosis to starting this study was 34 months (range 9–103 months), median number of prior therapies was 2 (range 0–6), median number of cycles on study was 1. 5 (range 1–7). Responses lasted for 4 months in 4 and a year in 1. The agent was tolerated in this population though some toxicities were notable: 2 (12%) had grade 2 rash (though many with grade 1), 5 with grade 2 (31%) and 1 (6%) grade 3 nausea, vomiting or diarrhea, 2 (12% with grade 2 and 1 (6%) with grade 3 liver function abnormalities, and 1 (6%) grade 4 myositis likely related to the agent. Unfortunately, many of the toxicities occurred shortly after initiating therapy, leading to early withdrawal from therapy before a full cycle and evaluation for response could be performed. Conclusions: Sorafenib was tolerated and provided some responses in this difficult to treat group of unselected patients. Due to many early withdrawals, the study was terminated early. Utility would be enhanced in future studies by devising methods to apriori select this targeted therapy for those most likely to respond. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1842 Perifosine (Keryx Biopharmaceuticals) is an oral alkylphospholipid that has been shown to have clinical activity in multiple myeloma and Waldenstrom's macroglobulinemia. Pre-clinical data suggest that its activity is due to inhibition of the Akt signal transduction pathway. The Akt pathway is known to be important for viability in CLL, another B-cell malignancy. Therefore, we investigated the in vitro activity of perifosine in freshly isolated primary CLL cells. CLL patients at the Duke University and Durham VA Medical Centers were enrolled in an IRB-approved research protocol for blood sample collection. CLL cells were negatively selected using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by flow cytometry. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined. We found the 50% effective dose (ED50) of perifosine for inducing cytotoxicity in CLL cells after a three-day incubation using the MTS colorimetric assay to be 510 nM (n = 29, range 120 – 1540 nM). CLL cells were obtained from patients with generally poor prognostic markers: 52% CD38+, 93% ZAP70+, 78% IgVH unmutated, 42% 17p deletion, 8% 11q deletion, 27% trisomy 12, 12% normal, and 12% 13q deletion as a sole abnormality. There were no statistical differences in ED50 between cells obtained from patients in high or low risk prognostic groups. Perifosine induced apoptosis in a dose- and time-dependent manner, measured by Annexin V and PI staining (n = 4). Based upon these pre-clinical results, we initiated a phase II study of perifosine (50 mg orally, twice daily) in relapsed or refractory CLL/small lymphocytic lymphoma (SLL) (NCT00873457). The primary objective of this study is to assess the response rate at 3 and 6 months of perifosine treatment in patients with relapsed or refractory CLL/SLL. Secondary objectives are to monitor toxicity, evaluate overall survival, progression-free survival and response duration, and perform laboratory correlates. Early interim results of this study are presented. Since trial initiation in September 2009, 13 patients have been enrolled. Nine patients had Rai stage III/IV disease at the time of therapy, and 4 patients were fludarabine-refractory. Patients had extensive prior treatment (median 4, range 1 – 11). Many patients had high-risk prognostic features: 9/11 IgVH unmutated, 10/13 CD38+, and 11/13 ZAP70+. Evaluation of interphase and metaphase cytogenetics demonstrated 4 patients with 17p deletion, two with 11q deletion, 2 with trisomy 12, 4 normal, and 4 with other complex cytogenetic anomalies. Of 12 patients who began therapy, 5 patients withdrew from the study prior to 3 months, 6 patients received at least 3 months of therapy, and 1 patient completed 6 months of therapy. Of the patients who received at least 3 months of therapy, there were 5 patients with stable disease, and one patient with partial response, using iwCLL response criteria. Grade 3/4 toxicities included anemia (n=2), fatigue (n=2), dehydration (n=1), febrile neutropenia (n=1), hyperbilirubinemia (n=1), hyponatremia (n=1), cough (n=1), and dyspnea (n=1). One patient required a dose reduction to 50 mg daily and two patients required dose delays due to toxicities. In conclusion, perifosine has potent in vitro activity against primary CLL cells. Preliminary results of this ongoing phase II study of oral perifosine in relapsed or refractory CLL/SLL demonstrate mostly disease stabilization in a group of very high-risk patients and an acceptable toxicity profile. Completion of this clinical study is necessary to determine if perifosine monotherapy has a potential role in the treatment of CLL/SLL. Disclosures: Sportelli: Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

Description: Background and Significance : Even though we have treatments for CLL, it remains an incurable leuke mia. We need new and better treatments for this disease. The Akt kinase is usually constitutively acti vated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. Chromosome deletion at 11q22–23 is frequently seen in CLL, and this cytogenetic abnormality portends a bad prognosis. The PPP2R1B gene that encodes the Ab constant regulatory subunit of the tumor suppressor protein phos phatase 2a (PP2a) is within the deleted segment in CLL patients with deleted 11q22–23. The resulting underexpression noted in those with deleted 11q22–23 leads to decreased PP2a activity in CLL cells. PP2a is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and nuclear factor kB (through IkK). We have developed apolipoprotein E-mimetic therapeutic peptides that potently decrease phosphorylation of Akt, decrease TNF and nitric oxide (NO) and NO synthase (NOS) expression, and display anti-inflammatory activity in vitro and in vivo. NOS is overexpressed in CLL, and its product NO inhibits CLL cell apoptosis. Methods : Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. The apoE-mimetic peptides were prepared by chemical synthesis. Results : The apoE-mimetic COG compounds are peptides of 10 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein. Samples from 6 early stage CLL patients and 11 normal individuals were examined. Five of 6 patients were Rai stage 0 at presentation, and one was stage 1. They had been followed 5.6 yr (median; range 1.9–10.6 yr). Five of 6 were CD38 negative, and 2 of 6 were Zap-70 positive. Of 5analyzed, all had mutated IgVH gene. Five of 6 patients had not been treated. Of 6 peptides examined, all displayed some cytotoxicity for CLL cells in vitro. Peptide COG 112 was the most potent, while the control peptide with an inverted sequence (COG 056) had very little or no activity (Table). CLL PBMC Agent ED50 (nM) ED50 range (n) ED50 (nM) ED50 range (n) COG 112 (active) 215 64 to 351 (6) 5,150 1,100 to 〉12,500 (6) COG 056 (control) 13,546 2771 to 20,418 (6) 〉25,000 20,470 to 〉25,500 (6) COG 112 induced cell death in a dose-dependent fashion in all patients’ samples.The ED50 of COG 112 for normal B cells was very high (〉 24,400 nM). COG 112 was approximatel 24 to 116 fold more potent for killing of CLL cells compared to normal PBMC or purified B cells. In vivo studies in normal mice using COG 112 revealed no toxicity even with doses of 100 mg/kg. Conclusions : ApoE mimetic peptides kill CLL cells in vitro with high efficacy and potency (ED50s in the low nanomolar range). The cytotoxicity is very specific for CLL cells compared to normal PBMC and B cells (24 to 116 fold more potent for CLL cells). Preliminary studies show that the peptide is nontoxic in vivo in normal mice. In vivo trials with apoE peptides in patients with CLL should help determine the toxicity and efficacy in patients.