We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. (Blood. 2006;108:3477-3483)

Panobinostat is a potent oral pandeacetylase inhibitor that leads to acetylation of intracellular proteins, inhibits cellular proliferation and induces apoptosis in leukemic cell lines. A phase Ia/II study was designed to determine the maximum-tolerated dose (MTD) of daily panobinostat, administered on two schedules: three times a week every week or every other week on a 28-day treatment cycle in patients with advanced hematologic malignancies. The criteria for hematologic dose-limiting toxicities differed between patients with indications associated with severe cytopenias at baseline (leukemia and myeloid disorders) and those less commonly associated with baseline cytopenias (lymphoma and myeloma). In patients with leukemia and myeloid disorders, 60 mg was the MTD for weekly as well as biweekly panobinostat. In patients with lymphoma and myeloma, 40 mg was the recommended dose for phase II evaluation (formal MTD not determined) of weekly panobinostat, and 60 mg was the MTD for biweekly panobinostat. Overall, panobinostat-related grade 3-4 adverse events included thrombocytopenia (41.5%), fatigue (21%) and neutropenia (21%). Single-agent activity was observed in several indications, including Hodgkin lymphoma and myelofibrosis. This phase Ia/II study provided a broad analysis of the safety profile and efficacy of single-agent panobinostat in patients with hematologic malignancies.

The bcl-2 oncoprotein, which is involved in the t(14,18) translocation, protects cells against apoptosis. We examined the effects of interferon-alpha (IFN-alpha) on bcl-2 protein expression and apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. None of 12 patients with B-CLL examined expressed the t(14,18) translocation; however, all these, and seven other patients, expressed significant levels of bcl-2 protein. In vitro, IFN-alpha (500 U/ml over 18 h) increased bcl-2 expression on CLL cells (to 200 +/- 23% of control MCF, as determined by indirect immunofluorescence and flow cytometry, n = 10, P < 0.001). All of eight patients who received IFN-alpha (3 megaunits subcutaneously three times a week) demonstrated an increase in bcl-2 expression on circulating malignant cells. CLL cells undergo apoptotic cell death when cultured in vitro (35.6 +/- 10.3% DNA fragmentation after 18 h, n = 10). In the presence of IFN-alpha, however, DNA fragmentation was reduced to 6.6 +/- 5.8% (n = 10, P < 0.001). IFN-alpha also protected CLL cells against apoptosis induced by hydrocortisone and gamma irradiation (reducing DNA fragmentation from 63.9 +/- 12.6% to 10.8 +/- 4.5% and from 80 +/- 2.9% to 5.4 +/- 1.6%, respectively, P < 0.001 for both). The protective effect of IFN-alpha was dose dependent, and maintained for up to 24 h. Our data demonstrate that bcl-2 expression and apoptosis of CLL cells can be influenced by cytokines. In addition, it seems unlikely that the observed clinical responses to IFN-alpha in patients with CLL are due to a direct effect on the malignant cells.

Romiplostim is an investigational Fc-peptide fusion protein (peptibody) that stimulates platelet production by a mechanism similar to endogenous thrombopoietin. MDS pts receiving hypomethylating agents such as azacytidine frequently develop clinically significant thrombocytopenia. This is an ongoing phase 2 multicenter, randomized, double-blind, placebo-controlled study evaluating the effect of romiplostim on the incidence of clinically significant thrombocytopenia in pts with low or intermediate risk MDS receiving azacytidine. Eligible pts were randomized (stratified by baseline [BL] platelet count [≥ or &lt;50 × 109/L]) 1:1:1 to 3 treatment groups to receive placebo or romiplostim at 500μg or 750μg weekly, by subcutaneous (s.c.) injection. All pts received 4 cycles of azacytidine 75mg/m2/day by s.c. injection for the 1st 7 days of each 28-day treatment cycle. The primary endpoint was the incidence of clinically significant thrombocytopenic events, defined as platelet counts &lt;50 × 109/L after Week 3 of treatment or receipt of platelet transfusions (txns) at any time during the treatment period. Secondary endpoints included the incidence of platelet txns and platelet nadir during azacytidine treatment cycles. Safety of romiplostim in combination with azacytidine was evaluated. Forty pts were randomized and treated. The 3 groups were balanced for the stratification factor, but some MDS disease characteristics were imbalanced including IPSS score &gt;1 (54%, placebo vs 23% and 29% romiplostim 500μg and 750μg). Results presented are from a planned interim analysis after all pts had completed or withdrawn from the treatment period. Overall pt incidence of clinically significant thrombocytopenic events was 85%, 62% and 71% for the placebo, romiplostim 500μg and 750μg groups, respectively. In a summary by azacytidine treatment cycle, the pt incidence of thrombocytopenic events per cycle was higher in the placebo group (50%–85% of pts) than in the romiplostim 500μg (44%–69% of pts) and 750μg (18%–64% of pts) groups, respectively. The pt incidence of platelet txns was 69% for placebo vs 46% and 36% for romiplostim 500μg and 750μg groups. Platelet counts on the 1st day of each azacytidine cycle and at the nadir of each 28-day cycle (Table) were lower in the placebo than the romiplostim groups. All pts (100%) had ≥1 adverse event (AE). Serious AEs (SAEs) were observed in 77%, 46%, and 71% of placebo, romiplostim 500μg and 750μg groups, respectively. Two pts in the romiplostim groups experienced ≥1 treatment-related SAE (1 arthralgia, romiplostim 500μg; 1 rash and hypersensitivity, romiplostim 750μg). Two pts in the placebo group had grade 3 or above bleeding events (1 pulmonary hemorrhage and 1 hemorrhage) vs 1 pt in the romiplostim 500μg group (epistaxis) and none in the romiplostim 750μg group. Two pts died in the placebo group (1 fungal pneumonia, 1 pulmonary hemorrhage) vs none in the romiplostim groups. One case of disease progression from MDS to AML was observed in the 500μg group. In summary, romiplostim reduced pt incidence of clinically significant thrombocytopenic events and platelet txns, and improved platelet nadir in MDS pts receiving azacytidine in this study. These findings are consistent with increased platelet counts over time. Romiplostim in combination with azacytidine appears to be well tolerated in this pt population. Azacytidine Treatment Cycle Placebo (N=13) 500μg Romiplostim (N=13) 750μg Romiplostim (N=14) n Cycle BLa Platelet nadirb N Cycle BLa Platelet nadirb n Cycle BLa Platelet nadirb aMedian of platelet count (×109/L) on 1st day of each azacytidine treatment cycle bMedian of lowest platelet count (×109/L) during each azacytidine treatment cycle BL = baseline 1 13 25 14 13 40 33 14 37 32 2 11 48 16 10 121 63 13 115 53 3 11 54 33 10 73 43 11 222 116 4 10 88 40 9 60 53 10 315 91

Low-dose IL-11 has activity in patients with BMF and is generally well tolerated.

Purpose Immunologic surveillance of minimal residual disease in chronic myelogenous leukemia (CML) may be relevant for long-term control or cure of CML. Little is known about immune-modulatory effects of nilotinib in vivo, potentially predicting response to therapy. Patients and Methods A prospective and comprehensive flow cytometry-based immunomonitoring program paralleled the ENEST1st clinical study, investigating 52 nilotinib-naïve patients with chronic-phase CML. Data were verified in independent validation cohorts. Results T cells of patients with CML at diagnosis expressed low l-selectin (CD62L) levels, which was not a result of proportional aberrations of T-cell subsets. Low numbers of CD62L-expressing CD4 and CD8 T cells correlated with higher Sokal score, increased spleen size, and high leukocyte and peripheral-blood blast counts. At month 6 during nilotinib therapy, CD62L expression returned to levels of healthy individuals. The level of CD62L loss on T cells directly correlated with the extent of soluble CD62L (sCD62L) elevation. In parallel, the proteolytic activity of tumor necrosis factor α-converting enzyme (TACE; ADAM17, CD156b), the metalloproteinase shedding CD62L, was increased at diagnosis and significantly decreased during nilotinib treatment. High CD62L expression on both CD4 and CD8 T cells and, vice versa, low sCD62L levels at CML diagnosis were linked to superior molecular responses. These findings were corroborated in independent validation cohorts. Conclusion We demonstrate the prognostic impact of CD62L shedding from T cells and increased sCD62L plasma levels at CML diagnosis on molecular response to tyrosine kinase inhibitor therapy in early chronic-phase CML. Functionally, decreased CD62L may be a consequence of increased TACE-mediated CD62L cleavage and potentially impairs immune-cell function. Larger prospective studies are ongoing to confirm the prognostic relevance of this finding.

Recent studies have shown that, when used in early stage disease, interferon-alpha (IFN-alpha) can produce a fall in the number of malignant cells in the peripheral blood of patients with B-CLL. In this study, we investigated the effect of IFN-alpha on natural killer (NK) cell and lymphokine-activated cell (LAK) activity in patients with B-CLL. In vitro, IFN-alpha (500 U/ml for 18 hours) induced LAK activity in patients with B-CLL (27.7 +/- 9.9%, n = 20), and IL-2 (500 U/ml for 5 days) produced similar activity (35.9 +/- 8.8%, n = 7). Despite the induction of LAK activity by IFN-alpha and IL2 in patients with B-CLL, the malignant cells remained resistant to both allogeneic and autologous LAK effectors. NK activity in patients with B-CLL is also low (23.1 +/- 7.2%, n = 20), and B-CLL cells were resistant to NK cell activity. In cold target competition assays, CLL cells did not compete with labelled K562 or Daudi targets in the NK and LAK assays, suggesting that the malignant cells are not recognised by the effector cells, and this may be related to low level of expression of the adhesion receptors, LFA-1 and ICAM-1. Finally, CLL cells were also resistant to antibody dependent cell mediated cytotoxicity, but were susceptible to antibody dependent complement mediated lysis. These results suggest that it is unlikely that the effects of IFN-alpha in B-CLL are due to the enhancement of NK or LAK activity.

Purpose The impact of proton pump inhibitors (PPIs) and histamine H2 receptor antagonists (H2 blockers) on the efficacy of nilotinib was evaluated. Methods Retrospective analyses were performed in patients with newly diagnosed Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia in chronic phase (CML-CP; N = 492) and in patients with imatinib-resistant or -intolerant Ph+ CML-CP (N = 256) treated with nilotinib. Results In the newly diagnosed population, 87 (17.7%) and 49 (10.0%) patients received PPIs and H2 blockers, respectively. Major molecular response at 12 months was achieved by 59 (49.6%) patients who received at least one PPI or H2 blocker (n = 119) and 153 (41.0%) patients who did not receive any comedication (n = 373; P = 0.13). PPIs and H2 blockers were used by 77 (30.1%) and 17 (6.6%) patients with imatinib-resistant or -intolerant CML-CP, respectively. Major cytogenetic response by 12 months was achieved by 55 (64.0%) patients who received at least one PPI or H2 blocker (n = 86) versus 98 (57.6%) patients who did not receive any comedication (n = 170; P = 0.40); 39 (45.3%) versus 65 (38.2%), respectively, achieved complete cytogenetic response by 12 months (P = 0.34). Similar findings were observed in patients who received comedication for >50% of the time on nilotinib therapy. Nilotinib steady-state trough concentration was not affected by the presence of PPIs or H2 blockers. Conclusions Concurrent use of PPIs or H2 blockers did not affect the pharmacokinetics and efficacy of nilotinib in patients with Ph+ CML-CP.