ALBERT

Description: MDS is a disease of the elderly marked by dysplasia of hematopoetic cell lines resulting in cytopenias and AML progression. There is currently no treatment approved to alter the natural history of the disease. Aberrant methylation associated with cancer is a potential target for pharmacologic therapy, and DacogenTM (decitabine) for injection (SuperGen, Inc.), a cytosine analogue, indirectly depletes methylcytosine after incorporation into DNA with subsequent inactivation of DNA methyltransferases. We report the results of a Phase III trial of decitabine (DAC) vs. supportive care (Supp.Care) in adult MDS patients with IPSS Intermediate (Int)-1 (31%), Int-2 (44%) and high risk disease (26%). Secondary MDS (14%) and previously treated (27%) MDS patients were not excluded. Bone marrows were assessed by a blinded, independent pathologist. 170 patients (accrued from July 2001 through April 2003 at 23 centers) were randomized 1:1 to either Supp.Care or DAC (a 3 hr infusion of 15mg/m2/hr every 8 hrs on 3 consecutive days every 6 wks for up to 10 cycles. The groups were comparable for numerous risk factors, including time from diagnosis (median 29 weeks for DAC and 35 weeks for Supp.Care). Kaplan Meier (KM) curves for time to AML progression or death showed early and clinically meaningful separation (consistent with clinical benefit) in favor of DAC in all patients (intent to treat; ITT), Int-2/High Risk, and High Risk patients. Using the Cox proportional hazards model for the ITT population, the probability of progression to AML or death was 1.68-fold greater for Sup.Care than for DAC (p=0.023). Median Time to AML or Death (Days) MDS group (n) DAC Supp.Care p-value 1Protocol specified test. 2Preferred test for analysis of early separation of KM curves. n=89 n=81 Wilcoxon1,2 Log rank1 All Patients (170) 338 263 0.046 0.204 Int-2/High Risk (118) 334 189 0.005 0.040 High Risk (44) 260 79 0.001 0.006 Treatment naïve (124) 354 189 0.005 0.034 Figure Figure Investigator reported response rate by International Working Group criteria was 25% (10% CR, 15% PR) for DAC vs. 0% for Supp.Care (p〈 0.001), with responses equally distributed across baseline subgroups. Time to response was 100 days and median duration was estimated at 〉9 months. Responders (CR and PR) vs. non-responders had a median survival of 678 days vs. 406 days (p= 0.038 Wilcoxon). There were no treatment related deaths. As expected, grade 3–4 toxicity (including hematologic toxicity, febrile neutropenia) occurred in more DAC patients than in Supp.Care patients. Most patients tolerated treatment well. DAC appears a promising therapy for MDS with manageable toxicity. Final results of independently reviewed response rates and clinical benefit (transfusion independence; Quality of Life) will be presented.

Description: DAC is a potent hypomethylating agent with clinical activity in patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). VPA is a histone deacetylase inhibitor used as an antiepileptic agent. In vitro, the combination of DAC with VPA results in synergistic antileukemia activity at doses of VPA above 1mM. Based on this data, we have developed a phase I/II study of this combination for pts with leukemia. The phase I of the study followed a classic 3+3 design. The dose of DAC was fixed: 15 mg/m2 iv daily for 10 days. This was based on a previous phase I study (Blood2004;103:1635) that indicated that this schedule had an optimal toxicity-response profile in this population. Three dose levels of VPA were selected: 20, 35 and 50 mg/kg. VPA was given orally for 10 days concomitantly with DAC. 22 pts have completed the phase I portion of the study (median age 56 years, range 4–78, 20 pts AML, 2 MDS). At dose level 1 (20 mg/kg of VPA) no grade III-IV toxicity was observed. At dose level 2 (VPA 35 mg/kg), 2 out 6 pts developed grade III neurotoxicity. Both pts were receiving high doses of other neurotropic agents. After IRB approval, 3 mores pts were treated at this dose level with no significant toxicity. Subsequently, 3 pts were treated at the highest planned dose level (50 mg/kg) with no toxicity observed. This cohort was then expanded to a total of 10 pts. One pt developed grade III neurotoxicity. No other severe drug-related toxicities were observed, but 5 patients at all dose levels developed grade II sedation/somnolence. Pancytopenia was induced in all pts. At dose level 1, one pt with refractory AML achieved complete remission (CR) after the second course of therapy. This is now maintained for 5 courses. At dose level 2, a patient with HIV disease and relapsed AML achieved CR after the third course of therapy, and 2 pts with relapsed AML achieved complete marrow responses (marrow blasts less then 5%, no recovery of peripheral counts). Of 3 pts evaluable for response at dose level 3, 1 pt with MDS has achieved CR after 1 course, and 1 with relapsed AML a complete marrow response. Median free VPA levels pretreatment were 0, and 25 mg/L on both days 5 and 10 and returned to 0 prior to next course. Histone acetylation measured by Western blot was observed in 3 pts (25%), all at doses above 20 mg/kg of VPA. Reactivation of p21 expression was induced in 4 out 11 pts analyzed. Global hypomethylation measured using a bisulfite PCR LINE assay was induced in 1 out 3 pts so far studied. Based on the toxicity observed, the phase II portion of the study was initiated. This is restricted to pts with AML/MDS. Seven pts have been accrued to this phase, and 8 out the 10 pts at dose level 3 of the phase I are also evaluable. The response data of this pts will be updated at the meeting. In summary, the combination of low dose DAC and VPA up to doses of 50 mg/kg can be safely administered to pts with leukemia although it may be complicated by neurotoxicity. Clinical and biological activity was observed at all dose levels.

Description: Several cytokines and growth factors are involved in the regulation of megakaryocytopoieses and platelet formation. Interleukin-11 (IL-11), IL-6, IL-3, IL-1b, and thrombopoietin (TPO) act synergistically to promote proliferation and maturation of megakaryocytes. Recombinant IL-11 and TPO are under clinical investigation as supplements to stimulate thrombopoiesis in patients with cancer. We investigated the plasma levels of TPO in 127 patients with chronic lymphocytic leukemia (CLL) and correlated these levels with platelet counts and various laboratory and clinical characteristics. TPO levels were significantly higher in patients with CLL (median, 232 pg/mL; range, 26.4–2714.5 pg/mL) than in healthy control subjects (P

Description: Prospective analysis of the importance of the plasma levels of β-2 microglobulin (B2M) in 553 patients with myelodysplastic syndrome (MDS) found that B2M is an independent prognostic variable for survival with weighted significance second only to the karyotype. The incorporation of the B2M covariate into risk assessment of MDS patients added significantly to the power of the IPSS to stratify MDS patients into risk categories. Our results further document that the 2 objectively measured covariates that display the highest power to predict survival, that is, karyotype and B2M, can alone be used for risk stratification. While the results must be verified in an independent and comparable population, our data strongly recommend routine measurement of B2M in patients with MDS.

Description: Janus kinases (JAK) are tyrosine kinases associated with both cytokine receptors and downstream signal transducer and activator of transcription (Stat) proteins. Upon activation of JAK by a variety of cytokines and growth factors, Stats translocate to the nucleus and promote transcription of target genes. Constitutive activation of Stat proteins in AML has been associated with poor prognosis and AG490, an inhibitor of this pathway, was shown to suppress AML cell proliferation in vitro. WP-1066 represents a further development of AG490 with biological activity at significantly lower concentrations. Therefore, we studied the effects of WP-1066 on the AML cell lines OCIM2 and K562 and on fresh bone marrow aspirates obtained from five newly diagnosed AML patients. We found that WP-1066 inhibited the proliferation of OCIM2 and K562 cells and of fresh marrow AML blast colony-forming cells in a dose-dependent fashion at concentrations ranging from 0.5 to 3 μM. WP-1066 completely abrogated the growth of leukemia cells at a concentration of 3 μM. Furthermore, WP-1066 induced a cell cycle arrest of OCIM2 and K562 cells. Incubation of AML cells with 2 μM of WP-1066 resulted in a time-dependent accumulation of OCIM2 and K562 cells in the sub-G0 phase of the cell cycle. Those leukemia cells underwent apoptotic cell death as assessed by annexin V-FITC. Incubation of OCIM2 cells with 0.5 to 3 μM WP-1066 for 2 hours induced a dose-dependent apoptosis in 52% of the cells. A 4 hour exposure of either OCIM2 or K562 cells to 2 μM of WP-1066 induced caspase 3 activation and PARP cleavage. As expected, WP-1066 inhibited Stat3 and Stat5 phosphorylation in K562 and OCIM2 cells both in a time- and dose-dependent manner, confirming that inhibition of the JAK-Stat pathway is its mechanism of action. Overall, our data showing that WP-1066 inhibits the JAK-Stat pathway, suppresses proliferation, induces cell cycle arrest and apoptosis of AML cells, suggest that the activity of this compound warrants further exploitation aimed at developing WP-1066 for future therapy of AML.

Description: RAD001 (everolimus, Novartis) is an orally bioavailable derivative of rapamycin with demonstrated anti-proliferative activity against a broad panel of tumor cell lines and antitumor activity in experimental animal models of human cancer. RAD001 inhibits the mammalian target of rapamycin (mTOR) signaling pathway, which is involved in regulating many aspects of cell growth and cell cycle progression. Several lines of evidence implicate the importance of the PI3K/Akt/mTOR pathway in hematological malignancies. As there has been extensive experience with this agent in the solid organ transplantation setting, two dose levels (5 and 10 mg/day) were evaluated in the Phase I portion of this study to determine the maximum tolerated dose (MTD) to be used in the Phase II portion. RAD001 was administered orally once a day at the starting dose level of 5 mg. Fifteen patients have been enrolled, of which 14 (3 B-CLL, 4 MDS, 1 MF, 1 NK/T cell lymphoma/leukemia, 1 MCL, 3 AML, 1 T-PLL) were evaluable for safety and toxicity. Median age was 65 years (range, 56 to 76 years). Twelve patients (86%) had received prior therapy (median, 2 regimens; range, 0 to 6 regimens). Median time on study was 29 days (range, 12 to 105 days). No dose-limiting toxicities were observed in the 6 patients enrolled in the Phase I portion of the study (cohorts of 3 patients per dose level). The most common adverse events were grade ≥ 2 and consisted of hyperglycemia, hyperlipidemia, hypocalcemia, hypomagnesemia, hypophosphatemia and hypokalemia, transaminitis, diarrhea, dermatitis, and mucositis. Grade 3 toxicities consisted of asymptomatic hypophosphatemia (2), and hyperglycemia (3). No patient experienced grade 4 toxicities or death from RAD001. Of the 14 evaluable patients, 2 patients with B-CLL had a 33% and 65% reduction in the size of lymph nodes, respectively, and 1 patient with MDS had decreased transfusion requirements. At the time of analysis, only 1 patient had discontinued therapy due to disease progression. These preliminary findings indicate that RAD001 is well tolerated at a daily dose of 10 mg/day and may have clinical activity in patients with hematologic malignancies. Enrollment is ongoing.

Description: Monitoring minimal residual disease (MRD) in chronic myeloid leukemia after imatinib therapy can help in determining therapeutic strategy. Sensitive methods based on the detection of the BCR/ABL fusion gene or its fusion transcript by FISH or RT-PCR, respectively, are used to monitor residual leukemia in peripheral blood or bone marrow cells. While sensitive, these methods require an invasive procedure to obtain bone marrow. The patchy nature of the residual disease may cause some of the reported variation in detecting MRD. We hypothesized that plasma RNA level reflects total body disease and is unaffected by the tendency of some myeloid cells not to circulate. We measured the BCR/ABL fusion transcript by real-time quantitative RT-PCR using RNA isolated from blood plasma. We used a quantitative RT-PCR assay capable of measuring both the b2a2 and b3a2 BCR/ABL fusion transcripts by targeting exon 13 of BCR with the 5′ and exon 2 of ABL by the 3′ primer. RNA isolated from 10–30 μl of frozen plasma was subjected to real-time quantitative RT-PCR using the Taqman technology. Quantification was achieved by extrapolation from a standard curve generated from a ten-fold dilution series of a synthetic oligonucleotide corresponding to the (+) strand of the target sequence. To normalize the fusion mRNA levels we also measured β-actin mRNA from the same samples. Assays were run in duplicate. The results of plasma RT-PCR were correlated with results of conventional cytogenetic analysis, FISH, and quantitative RT-PCR of bone marrow suspension (“bone marrow PCR”). Plasma and bone marrow samples were obtained at 3, 6, 9, 12 and 15 months after imatinib therapy. Ninety-five plasma samples were analyzed. There was a strong positive correlation between the results of plasma RT-PCR vs. positive cytogenetics (n=95, p