Addition Of Imatinib Research Articles

Turn-off fluorescence of nitrogen and sulfur carbon quantum dots as effective fluorescent probes for determination of imatinib. Application to biological fluids

Nitrogen and sulfur carbon quantum dots(N,S-CQDs) as effective fluorescent nanoprobes were synthesized through one-step-hydrothermal method using thiosemicarbazide (as nitrogen and sulfur source) and citric acid (as carbon source). The highly fluorescent N,S-CQDs were subjected to various characterization techniques. The fluorescence of the synthesized N,S-CQDs is characterized by maximum fluorescence emission at 415 nm after excitation at 345 nm and a high quantum yield of 0.58. The native N,S-CQDs fluorescence is quantitatively quenched upon addition of imatinib (IMA), so they are used for its spectrofluorimetric determination in its pharmaceutical formulations and biological fluids. Under optimal conditions, N,S-CQDs exhibited a “turn-off” fluorescence response to IMA over the range of 1.0 to 15.0 µg/mL with a limit of quantification of 0.42 µg/mL and a lower detection limit of 0.14 µg/mL. Stern-Volmer equation was used to study the mechanism of quenching and it was found to occur through static quenching mechanism. The method was extended to the in-vitro determination of the drug in spiked human urine and plasma samples and the percent recoveries were satisfactory.

PCN137 - THE ECONOMIC EVALUATION OF IMATINIB COMBINED WITH CHEMOTHERAPY FOR PEDIATRIC PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA IN CHINA

Pediatric Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) is a very rare disease with poor prognosis. imatinib combined with chemotherapy is currently the first-line treatment. However, Ph+ ALL has long been unlisted as the relevant indication for imatinib in the national healthcare reimbursement system in China. To inform the relevant decision makers, we aimed to evaluate the cost-effectiveness of imatinib combined with conventional chemotherapy (imatinib+CC) as compared to conventional chemotherapy alone (CC) for this neglected and vulnerable population. We first conducted a systematic review to summarize literature evidence on clinical effectiveness and cost-effectiveness of imatinib+CC, and then developed a Markov model based on the best available published data and local expert opinions. From the perspective of the Chinese healthcare system, the model simulated the following health states over a life-time horizon for a hypothetical cohort of newly diagnosed 8-year old Ph+ ALL children: event-free survival, relapsed/resistant disease, cure, and death. A few of tunnel states were also built to reflect the treatment pathway according to the clinical guideline CCLG-ALL 2008 (HR protocol). In the absence of published cost data for Ph+ ALL patients, we sought expert opinions from an ongoing Chinese Childhood Cancer Group ALL-2015 trial consisting of 20 centers nationwide. The base case analysis showed quality-adjusted life-years (QALYs) increased by 1.25 QALYs with imatinib in addition to CC. The resulting mean Incremental Cost-Effectiveness Ratio (ICER) was CNY 32,725/QALY. At a willingness-to-pay threshold of CNY 53,935/QALY according to China’s GDP per capita in 2016, imatinib+CC was considered highly cost-effective. The decision outcomes were robust to the probabilistic and deterministic sensitivity analyses. For children with Ph+ ALL, imatinib+CC was found to be a cost-effective intervention, compared to CC alone. The addition of imatinib is associated with increased life years and increased QALYs at a reasonable incremental cost.

A Treatment Protocol with Imatinib and Intensive Chemotherapy for Pediatric Philadelphia Positive Acute Lymphoblastic Leukemia Patients: A Single-Arm, Intergroup Study (EsPhALL 2010-2014)

▪Introduction. Approximately 3-5% of pediatric ALL patients present with the Philadelphia chromosome (Ph+). In the past, Ph+ ALL was associated with a poor prognosis, with long-term event-free-survival (EFS) rates of approximately 30% and most patients allocated to hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1). The COG AALL0031 study showed that continuous high imatinib exposure improved outcome with no increase in toxicity and that HSCT offered no advantage over intensive chemotherapy plus imatinib (Schultz et al, JCO2009). Contemporarily, the EsPhALL 2004-2009 intergroup study of post-induction treatment of Ph+ ALL randomly tested the discontinuous use of imatinib added to BFM ALL high risk chemotherapy after the Induction phase in patients with early response, showing an approximate 10% advantage in long-term DFS. All non-responding patients received imatinib and overall 80% received HSCT in CR1 (Biondi et al, Lancet Oncology 2012). Based on both studies, in 2010 the EsPhALL trial was amended into a single arm study to give all patients, on top of the BFM ALL high risk chemotherapy, imatinib 300/mg/m2/day continuously since day 15 of Induction and to gradually decrease the use of HSCT. We present here the results of this EsPhALL 2010-2014 study.Methods. Ph+ ALL patients aged 1-17 years were enrolled by 10 national study groups (AIEOP, BFM-G/CH, COALL, FRALLE-EORTC, NOPHO, MRC, DCOG, CPH, PINDA, and HKPHOSG), mainly in Europe. MRD was assessed by PCR using IG/TR targets or BCR/ABL1 in hierarchical order, according to Euro-MRD rules. Eligibility to HSCT depended on early response and, after 2012, on MRD at end of Protocol IB (≥ 5 x 10-4) and at the end of consolidation block 3 (any positivity in patients with MRD< 5 x 10-4 at end of IB). Imatinib was recommended throughout the first year after transplant. Patients who continued on chemotherapy received treatment until 24 months after diagnosis.Results. Overall 158 patients were registered from January 2010 to December 2014; 3 were not eligible, leaving 155 evaluable patients. Sixty-two patients (40%) were ≥ 10 years, 73 (47%) had ≥50,000 WBC count at diagnosis, 103 (84%) had p190. 102 (66%) patients achieved both early good response and CR1 at end of IA and were classified as good risk, while the remaining 53 (34%) were at poor risk. CR1 was achieved by 151 patients (97%) at the end of Induction and by the remaining 4 at the end of IB. Overall, 59 patients (38%) underwent HSCT in CR1. Nineteen patients discontinued treatment, 17 during chemotherapy, 15 due to serious adverse events (SAE, of which 9 related to imatinib - mainly infections) and the remaining 2 to MRD increase (1) and to transfer to adult unit (1). Among patients who underwent HSCT in CR1, 2 discontinued imatinib after HSCT, due to MRD increase (1) and gastrointestinal toxicity related to imatinib (1). Overall, 185 SAE were observed in 86 (55%) patients. The most frequent SAE was infection which occurred in 50 (32%) patients. Other toxicities included osteonecrosis (7 patients, 5%) and gastrointestinal disorders (9 patients, 6%). On the complete cohort of 155 patients, with a median follow-up of 57 months (range 1-86), the 5-year EFS and survival were 57.0 (SE 4.1) and 71.8 (SE 3.8), respectively. The 5-year cumulative incidence of relapse was 26.9 (SE 3.7). Overall, 65 events occurred, 46 in 96 patients who received chemotherapy only (32 relapses and 14 deaths in CCR) and 19 in 59 HSCT in CR1 (8 relapses and 11 deaths in CCR). Overall, relapses occurred in the BM (22), BM+CNS (11), BM+eye (1) and CNS (6). All deaths in CCR, except for 1 in chemotherapy and 3 after HSCT, were due to infection. Half of the deaths in CCR (12/25) occurred in 2 unexpected clusters, in HSCT performed in 2010-2011 and in chemotherapy-only patients ≥ 10 years. Further investigations did not reveal any common pattern within the clusters.Conclusions: Addition of imatinib given continuously since day 15 of induction markedly increased CR rate to 97% from 50% of the EsPhALL 2004-2009 study. Moreover, in EsPhALL 2010-2014 only 38% of patients underwent HSCT in CR1 (decreasing from 56% in 2010 to 28% in 2014), compared with 81% in the previous study. In spite of this marked decrease in HSCT, the outcome was similar in the 2 studies: 5-year EFS and survival were 57.0% and 71.8% versus 60.3% and 71.6%, respectively in EsPhALL 2010-2014 versus EsPhALL 2004-2009. DisclosuresCazzaniga:Fondazione Tettamanti onlus: Employment; Italian Association for Cancer Research: Research Funding. Saha:Shire: Research Funding. Schrappe:Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.

PDGFR Inhibitor Imatinib May be a Potent Drug in Treating Essential Thrombocythemia

Approximates 80% of essential thrombocythemia (ET) patients carrying JAK2, CALR, MPL mutations. However, there is still 20% of ET patients are negative for all three mutations. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) and its receptor (PDGFR) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis. Therefore, we speculated that PDGF/PDGFR may plays an important role in megakaryocyte/platelet related disease, such as ET. In this study, we found an increase of PDGF-BB and PDGFR expression in ET patients comparing to the normal persons. We then used the adjusted dosage of PDGF-BB (1.5ug/kg/d) to establish an ET mimic mouse model. We found that PDGF-BB can enhance platelet production in vivo significantly, while PDGFR inhibitor imatinib can block this effect. Bone marrow histology data also confirmed the megakaryopoiesis effect of PDGF, as shown by an increased number of megakaryocytes in PDGF-treated mouse. The addition of imatinib can block the effect, evident by a decrease number of megakaryocytes and an increase of apoptosis. Furthermore, we demonstrated that PDGF-BB activated theP-JAK2, the P-STAT3 and the P-AKT expression in the megakaryocytic cell line CHRF-288, while addition of imatinib can reduce the phosphorylation level of all three genes. Our results suggested that the overexpression of PDGF-BB and PDGFR may be important in the pathogenesis of ET, especially in the JAK2-mutation negative ET. The elevated PDGF-BB activates PDGFR with subsequently activation of the JAK2/ STAT3 and PI3K/AKT pathway. Imatinib may have an effect on treating ET via blocking PDGFR and the following JAK2/STAT3 and PI3K/AKT pathway. This study suggests that PDGF/PDGFR may be involved in the pathogenesis of essential thrombocythemia, and the expression of PDGF-BB and PDGFR is potential to be a diagnostic index of ET. Blockage of PDGFR by imatinib may be a new therapeutic target for ET. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of a New AHI-1-Mediated-PP2A-Beta-Catenin-BCR-ABL-JAK2 Complex Modulating Resistance of CML Stem/Progenitor Cells to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of early phase CML patients. However, TKI monotherapies are not curative and initial and acquired resistance remain challenges. Particularly, leukemic stem cells (LSCs) are less responsive to TKIs and are a critical target population for TKI resistance. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is highly upregulated in LSCs and interacts with multiple kinases, including BCR-ABL and JAK2. We further showed that AHI-1-mediated complexes contribute to enhanced transforming activity of BCR-ABL and drug resistance of LSCs, suggesting AHI-1 as a new therapeutic target in CML. By screening the Prestwick Chemical Library, we have recently identified a specific growth inhibitory compound that potentially targets AHI-1: Cantharidin (CAN), an inhibitor of protein phosphatase 2A (PP2A). PP2A is a family of serine/threonine phosphatases that regulate cell signaling cascades involved in proliferation and cell cycle control of cancer cells. Evidence suggests the dysfunction of specific PP2A protein complexes is primarily responsible for the changes that lead to cell transformation. It was also reported that PP2A activity is downregulated in CML cells, particularly in blast crisis CML, due to overexpression of SET, and that the use of a PP2A activator inhibits the growth of CML cells. Interestingly, we have now demonstrated that CAN inhibits the growth of AHI-1-transduced BCR-ABL+ cells by about 30% compared to control cells, but this effect was significantly enhanced to 93% with the addition of imatinib (IM). As well, AHI-1-suppressed cells were more sensitive to CAN treatment. PP2A is highly expressed in K562 and IM-resistant K562 cells and combination treatment with CAN and TKI prevents growth and induces apoptosis in these cells more effectively than single treatments (2-3 fold, p<0.001). The combination also greatly reduced colony formation (CFC) of CD34+ CML cells, but CAN also inhibited CFCs of CD34+ normal bone marrow (BM) cells. To overcome toxicity issues, we have recently evaluated new, pre-clinically validated PP2A inhibitors, LB100 and LB102. These specifically suppress PP2A activity up to 75% in CML cells, as determined by immunoprecipitation (IP) phosphatase assays, and inhibit growth of these cells. Importantly, PP2A inhibitors plus TKIs significantly reduced the yield of colonies from CD34+ CML stem/progenitor cells compared to any single agent or combination of TKIs and this enhanced effect was most pronounced on cells from IM nonresponders (p<0.05). New PP2A inhibitors are also much less toxic to CD34+ normal BM cells compared to CAN (2-3 fold, p<0.001). Long-term culture-initiating cell assays showed that more primitive cells were eliminated to a greater extent by combination treatments. Moreover, the combination resulted in strong synergy in IM-insensitive cells (CI value <0.5). Notably, cell cycle analysis showed that treatment with LB100/LB102 alone induces a shift from G1 to G2/M phase (3-fold, p<0.05). A similar shift in the cell population was observed after combination treatment with IM, suggesting that the G2/M phase arrest is solely due to PP2A inhibition. Confocal microscopy showed the G2/M arrest, leading to mitotic catastrophe. Mechanistically, we have identified the PP2A subunit B (PR55a) as an AHI-1 interacting protein using IP/mass spectrometry; this was further confirmed in AHI-1-transduced cells by IP-Western analysis. Most interestingly, Western blot analysis showed that the combination treatment not only inhibits tyrosine phosphorylation of BCR-ABL, JAK2, STAT5, AKT and P38, but also significantly inhibits protein expression of these proteins compared to single agents. Inhibition of protein expression of AHI-1 and β-catenin by the combination was also observed and a new protein interaction between AHI-1 and β-catenin was identified. Moreover, the combination directly affects PP2A-mediated β-catenin dephosphorylation and BCR-ABL-mediated phosphorylation of β-catenin in CML cells, which leads to protein degradation of multiple proteins. In summary, we have uncovered new AHI-1-PP2A (PR55a) and AHI-1- β-catenin interactions and simultaneously targeting both BCR-ABL and PP2A activities in CML LSCs may offer an important new therapeutic possibility, through destabilization of the protein-protein interactions mediated by AHI-1. DisclosuresNo relevant conflicts of interest to declare.

Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. DisclosuresNo relevant conflicts of interest to declare.

Nanograting structure promotes lamellipodia-based cell collective migration and wound healing.

Wound healing is a dynamic and complex process of replacing missing or dead cell structures and tissue layers. The aim of this research is to discover biocompatible materials and drugs that can promote cell migration in the wound area and thus enhance desirable wound healing effects. In this paper, we report that PDMS nanogratings could accelerate the migration of epithelial cells along the grating axis, and the addition of Imatinib could further increase the epithelial cell wound healing speed to 1.6 times the speed of control cells. We also demonstrate that this migration is mediated by lamellipodia protrusion, and is Rac1-GTPase activity dependent. Lastly, we discuss the potential application and prospect of different nanostructured biomaterials for wound healing studies.

Therapeutic superiority of a TCR-like antibody to an intracellular WT1 oncogene peptide compared with the tyrosine kinase inhibitor imatinib in a mouse model of Philadelphia chromosome positive (Ph+) ALL.

3047 Background: Acute and chronic leukemias demonstrate significantly increased expression of the Wilms tumor gene 1 (WT1) product, including CD34+ CML stem cells, making WT1 an attractive therapeutic target. However, WT1 protein is intracellular and currently un-druggable. ESKM is a fully human IgG1 antibody that targets a 9 amino acid sequence (RMF) of the protein WT1 in the context of HLA-A0201. Methods: BV173 is an HLA-A0201 positive Ph+ ALL cell line. It over-expresses WT1 and binds strongly to ESKM. We evaluated the in vitro and in vivo efficacy of ESKM in combination with TKIs. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. In vivo tumor growth was assessed in NSG mice bearing disseminated luciferase tagged BV173 with bioluminescence imaging and flow cytometry of the bone marrow after sacrifice. Imatinib was used at maximum tolerated doses for these mice as determined in pilot studies. Results: The addition of imatinib in vitro did not affect the ability of ESKM to perform ADCC. The BV173 engrafted NSG mice treated with ESKM with and without TKIs showed tumor regression one week after beginning therapy, clearing leukemia from the liver and spleen. Mice relapsed primarily in the bone marrow, with increasing luciferase signal after two weeks of therapy. Compared to untreated control animals, after 5 weeks of therapy, imatinib alone only reduced tumor growth by 45%; ESKM alone reduced growth by 81%, and the combination of ESKM and imatinib reduced growth by more than 95%. Flow cytometry of cells remaining after treatment showed binding of ESKM, suggesting escape was not due to down regulation of the epitope. Conclusions: In this mouse model of Ph+ ALL, ESKM antibody therapy is superior to imatinib and the combination of both modalities is additive. This antibody is efficacious in vitro and in vivo against WT1 overexpressing leukemias, in context of HLA-A0201. This combination holds promise as a therapy for leukemias in patients who are HLA-A0201 positive, with the potential of improved cytoreduction in patients with Ph+ leukemias.

Complex effects of imatinib on spontaneous and oxytocin-induced contractions in human non-pregnant myometrium

Human myometrium includes two important cell populations involved in its contractility: smooth muscle fibers and interstitial cells. The pacemaking mechanism is not yet identified, but it is possible that myometrial smooth muscle cells contract in response to a signal generated by c-kit positive interstitial cells. The aim of this study was to investigate the effects of imatinib as a c-kit receptor antagonist on the spontaneous or oxytocin (OT) induced contractions of human non-pregnant myometrium in vitro. Myometrial strips were obtained from non-pregnant women (reproductive age) undergoing hysterectomy for benign indications. The strips were suspended in organ baths for recording of isometric tension. Imatinib effects were assessed on spontaneous contraction and after preexposure to OT.Direct exposure of myometrial strips to imatinib inhibits both amplitude and frequency of contractions (80-320 μM) in a dose dependent manner. Amplitude reverted back to 90% of the baseline amplitude by consequent addition of imatinib (until 480 μM). Total inhibition of myometrial contraction was obtained after addition of OT 60 nM. If myometrium was pre-exposed to OT (320 nM), imatinib 80-160 μm increased amplitude, while decreasing frequency. These data provide evidence that telocytes may be involved as modulators of the spontaneous contractions of the non-pregnant human uterus, via a tyrosine-kinase independent signaling pathway.

Wnt-Pathway Directed Compound Targets Blast Crisis and Chronic Phase CML Leukemia Stem Progenitors.

Abstract 2168Poster Board II-145The deregulated tyrosine kinase associated with t(9;22) fusion protein BCR-ABL is efficiently targeted by tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib). It is generally believed that most patients have residual disease. The inability to eliminate the refractory leukemia stem cell in chronic myeloid leukemia (CML) is not well understood. The refractory stem cell is present despite effective inhibition of the BCR-ABL kinase in these cells, and dissociation between kinase inhibition and cell death implies lack of dependency on BCR-ABL aberrant kinase activity, unlike the one seen in more mature CML cells. Thus the oncogene addictive dependency of these leukemia stem cells may be overcome by intrinsic and/or extrinsic factors/pathways. One such pathway involved in hematopoeitic stem cell maintenance is the Wnt/β-catenin signaling pathway. BCR-ABL signaling is known to directly activate the Wnt/β-catenin pathway. The loss of function of a negative regulator of the Wnt/β-catenin pathway, known as GSK3β is associated with CML progression from chronic phase to the blast phase. Lastly, the loss of β-catenin in murine models impairs the self renewal capacity of both the normal and the BCR-ABL+ leukemia stem cell. Thus this pathway is important during various stages of the disease.We explored the effect of Wnt inhibition using a novel Wnt pathway inhibitor (AG-214, University of Michigan). AG-214 is structurally related to a previously reported Wnt-inhibitor. AG-214 is able to antagonize β-catenin/TCF in luciferase reporter assays, and expression of Wnt targets in colon cancer cell lines. We utilized a serum free culture system with 5 added cytokines and treated both CML blast crisis CD34+ as well as chronic phase CD34+ cells with AG-214. Our in vitro experiments show that primary blast crisis CD34+ cells are induced to undergo apoptosis at an IC-50 of approximately 2 μM. Furthermore, combination of 1.25 μM of AG-214 with 2 μM Imatinib achieved greater than 50% apoptosis. Analysis of chronic phase CML CD34+ cells showed that targeting of Wnt/β-catenin pathway requires higher concentration of the Wnt-inhibitor (>2.5 μM), and that the addition of Imatinib can cooperate to enhance the apoptotic response. Furthermore, chronic phase progenitors (Lin-CD38+/CD34+) are more sensitive to lower concentrations of AG-214 whereas 5 μM is required to induce significant apoptosis of ∼70% in the primitive leukemia stem cell (Lin-/CD38-/CD34+) population, and addition of 2 μM Imatinib increased their apoptotic response to ∼84%. Normal CD34+ cells do not undergo significant apoptosis with 5 μM of AG-214 (∼10%) even when combined with 2 μM Imatinib (∼20%). Targeting of the Wnt/β-catenin pathway enhances apoptosis in both blast crisis and chronic phase CML progenitors and leukemia stem cells. Disclosures:No relevant conflicts of interest to declare.

Treatment with Tyrosine Kinase Inhibitors May Impair the Potential Curative Effect of Allogeneic Stem Cell Transplantation.

Abstract 857▪▪This icon denotes an abstract that is clinically relevant.Tyrosine kinase inhibitors (TKI) like imatinib and dasatinib are the current treatment of choice for patients with chronic myeloid leukemia (CML). Although most patients enter a complete remission during treatment, cure of the disease is usually not achieved since recurrence of the disease is seen in the majority of patients upon discontinuation of the treatment, indicating that the leukemic stem cell is not efficiently targeted. Furthermore, in accelerated phase and blast crisis of CML TKI treatment only results in temporary control of the disease. In these situations allogeneic stem cell transplantation (allo-SCT) and application of donor T cells may be the only curative treatment. Besides the direct anti-leukemic effect of allo-SCT, alloreactive T cells recognizing CML (progenitor) cells, and the formation of immunological memory may lead to effective lifelong immune surveillance. Therefore, we investigated whether the leukemic cells persisting during TKI treatment are susceptible targets for the anti-leukemic effect mediated by donor T cells after allo-SCT and whether continuous TKI treatment may have an additive effect during the immunological intervention. To investigate the anti-leukemic effect of the two strategies, CD34+ positive CML cells were isolated from bone marrow, and labeled with the fluorescent dyes CFSE or PKH to allow monitoring of single cell proliferation. CML cells were exposed to imatinib (1-100μM) or dasatinib (0.01-50nM), and/or to CD8+ alloreactive cytotoxic T lymphocyte (CTL) clones in the presence of proliferation-inducing cytokines. The number, phenotype, and proliferative status of the CML cells persisting after single and combined interventions were measured by quantitative flowcytometric analysis. In the absence of therapeutic interventions the majority of CD34+ CML cells entered proliferation. However, a small population of CD34+ CML stem cells residing in the non-dividing peak could be identified despite the addition of cytokines. Addition of imatinib or dasatinib resulted in efficient dose-dependent induction of cell death of the leukemic cells (99% lysis by 25μM imatinib or 10nM dasatinib). However, the population of quiescent CD34+ CML stem cells was not affected. Moreover, the number of cells present in the non-dividing population increased 2-fold compared to the non-treated controls at the highest TKI concentrations, indicating additional growth arrest of a population of proliferating CML precursor cells. We next tested the capacity of different HLA-A2-restricted CD8+ CTL clones to kill non-treated or imatinib or dasatinib treated CML cells. Whereas the proliferating CD34+ CML precursors were efficiently lysed, the population of quiescent stem cells was capable of withstanding CTL exposure. Detailed phenotypic analysis revealed significant downregulation of HLA-A2 and the adhesion molecules CD49d and CD58 on these quiescent cells, probably resulting in the impaired ability of these target cells to form a high avidity interaction with the T cells. The increased population of non-dividing cells as a result of the TKI pretreatment showed similar resistance to T cell induced cell death, indicating that TKI treatment may even diminish the anti-leukemic effect of allo-SCT. In the absence of therapeutic control, as mimicked by the removal of T cells and TKI from the cultures, outgrowth of the leukemic cells re-occurred, illustrating their capacity to give rise to a relapse of the disease. Next, we analyzed the effect of TKI treatment on T cell survival and functionality. Whereas resting primary T cells were insensitive to TKI treatment, T cells activated by either polyclonal stimulation with anti-CD3/CD28 beads or stimulation with allogeneic stimulator cells died after TKI exposure at similar concentrations as the leukemic cells. In conclusion, TKI treatment results in selection of a population of quiescent leukemic stem cells showing cross-resistance to CTL-induced cell death, most likely due to their inability to form a high avidity interaction. Moreover, T cells actively participating in the anti-leukemic immune response after allo-SCT are suppressed by TKI. These data indicate that continuous TKI treatment may potentially hamper the curative effect of allo-SCT. Disclosures:No relevant conflicts of interest to declare.

Treatment of Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma (ALL/LL). Results of a Single Center.

Abstract 4108 IntroductionALL/LL is a heterogenous disease with various subtypes that differ markedly in their cellular and molecular characteristics. Chemotherapy regimens, response to therapy and risk of relapse also vary among different disease subtypes. Here we report the experience in our center in the management of this heterogeneous group of haematological malignancies. ObjectivesThe objectives of the study are:- To determine the event free survival (EFS) in patients diagnosed with different subtypes of ALL/LL, including those who received an allogeneic stem cell transplantation.- To determine the overall survival (OS) for the patients diagnosed of ALL/LL and for those who received an allogeneic stem cell transplantation. Patients and Methods:Forty-two patients have been diagnosed of ALL/LL in our hospital from February'02 to April'09. 24 male (57%) and 18 female (43%), median age of 42,5 years (18-74), including sixteen cases of B-ALL (38%); 13 Ph+ ALL (31%); 6 T-ALL (14%) and 7 LL (17%).B and T-ALL were treated with the same chemotherapy regimen (Pethema: Spanish group for the treatment of haematological malignancies). Ph+ ALL were treated with chemotherapy and the addition of Imatinib (Pethema regimen) and Lymphoblastic Lymphomas (LL) were treated with HyperCVAD regimen. Elderly patients or those with significant comorbidities were treated with “adapted risk” chemotherapy regimens. Allogeneic stem cell transplantation, related or unrelated, was indicated in first complete remission (CR) for all cases of Ph+ ALL, and cases of B or T ALL who do not achieved CR with negative Minimal Residual Disease (MRD), or in second CR for cases with LL. ResultsThirty-three patients (78%) achieved CR at the end of the first induction phase. Two patients (5%) died in this phase and 5 patients (14% of the evaluable patients) relapsed at any moment of the treatment scheduled for them. Fourteen patients (33%) received an allogeneic transplantation: 7 (50%) from an unrelated donor. Nine of this 14 patients (64%) were transplanted in 1st CR. At the moment of the study, 21 patients (50%) are alive.The EFS for the patients included in the study is 41% with a median follow-up of 10,47 months (1,1-78,1). The EFS for the different subtypes are: 57% for B-ALL; 23% for Ph+ ALL; 44% for T-ALL and 57% for LL. The EFS for the transplanted patients is 34% and for the non transplanted patients group is 49%The OS is 43% with a median follow up of 11,57 months (1,11-78,1). The OS for the transplanted patients is 40% and for the rest of the patients is 48% ConclusionsPethema chemotherapy regimens for B, T and Ph + ALL, achieve a good percentage of CR, according to other similar regimens of chemotherapy reported. However, toxicity and relapses are still elevated. The research of a related or unrelated HLA-identical donor must be done from the diagnosis, specially in those forms of recognized bad prognosis. Disclosures:No relevant conflicts of interest to declare.

Preadipocyte apoptosis is prevented by macrophage-conditioned medium in a PDGF-dependent manner

Obesity is associated with macrophage accumulation and inflammation in adipose tissue. Macrophage-secreted factors have been reported to inhibit the differentiation of preadipocytes into adipocytes and to modulate adipogenic extracellular matrix gene expression. To enlarge our understanding of macrophages and the scope of their interactions with preadipocytes, we investigated their effect on preadipocyte survival. Acute exposure of 3T3-L1 preadipocytes to J774A.1 macrophage-conditioned medium (MacCM) stimulated platelet-derived growth factor receptor (PDGFR) tyrosine phosphorylation by 4.1-fold. There were significant increases in the phosphocontent of downstream PDGFR targets Akt and ERK1/2 (5.3-fold and 2.4-fold, respectively) that were inhibited by PDGF immunoneutralization or by the selective PDGFR inhibitor imatinib. Serum-free J774A.1-MacCM or RAW264.7-MacCM completely prevented 3T3-L1 preadipocyte apoptosis normally induced by serum deprivation. Addition of PDGF alone to serum-free control medium was sufficient to prevent 3T3-L1 preadipocyte apoptosis. Inhibition of PDGFR activation by MacCM, either by addition of imatinib or by PDGF immunodepletion of MacCM, effectively disrupted the prosurvival effect. In summary, our data indicate that MacCM promotes preadipocyte survival in a PDGF-dependent manner.

Regulation of FLT3 and its ligand in normal hematopoietic progenitor cells

FLT3 and its ligand (FL) are one of the regulators of normal hematopoiesis. Ligand-independent activation of FLT3 occurs in about 30% of acute myeloid leukemia cases and is one goal for selectively targeted therapies. However, the function of FLT3/FL in the regulation of non-malignant immature hematopoietic cells is poorly characterized. In order to elucidate the role of FLT3 in normal hematopoiesis, human adult CD34(+) hematopoietic progenitor cells were cultured in cytokine-supplemented liquid culture in the presence or absence of FLT3 inhibition by CEP-701 (lestaurtinib). Total cell number, lineage-committed, and primitive progenitors and apoptosis were assayed. FLT3 expression and FL secretion in various conditions were analyzed by fluorescent activated cell sorter and enzyme-linked immunosorbent assay. Effects of nonspecific targeting of FLT3 were evaluated with addition of imatinib (Gleevec) to cell cultures. It is demonstrated that FLT3 inhibition impaired cell and progenitor cell growth and increased the rate in apoptosis. Effects were observed independent of addition of FL. The dose-dependent growth inhibition was partially equalized by inhibiting FL with a neutralizing antibody. FLT3 inhibition resulted in markedly increased production of FL by cultured CD34(+) cells as well as upregulation of FLT3 expression. Imatinib mimicked effects of selective FLT3 inhibition. In conclusion, FLT3 and its ligand regulate proliferation of hematopoietic progenitor cells in an autocrine/paracrine manner Nonspecific inhibition of FLT3 may contribute to hematotoxicity caused by imatinib treatment.

Single Center Experience with Philadelphia Chromosome-Positive Acute Myeloid Leukemia.

Background: Rare instances of Philadelphia chromosome-positive (Ph+) acute myeloid leukemia (AML) have been reported, constituting <1% of de novo cases. However, differentiating these cases from chronic myeloid leukemia presenting in blast phase (CML-BP) has proven difficult. Several clinical and pathologic criteria have been proposed to distinguish Ph+ AML from CML-BP, including absence of an antecedent hematologic disorder, lack of evidence of a chronic or accelerated phase of CML after induction therapy, infrequent splenomegaly and peripheral eosinophilia or basophilia, and bone marrow characteristics such as lower cellularity, basophilia, and myeloid:erythroid ratio.Methods: We searched the M.D. Anderson Cancer Center leukemia database to identify all pts that had been diagnosed with Ph+ AML by cytogenetic analysis, between 1980 and 2006. Clinical, laboratory, and hematopathologic data were reviewed in order to separate them into the following groups: Ph+ AML, CML-BP, acute biphenotypic leukemia, and indeterminate. The following scoring system was employed: 1 point (splenomegaly), 1 point (peripheral eosinophilia and/or basophilia), 1 point (bone marrow basophilia or additional copy of Ph+ chromosome or trisomy 8 or isochromosome17). Zero points was classified as Ph+ AML, 1 point as indeterminate, and 2–3 points as CML-BP. If there was evidence of an antecedent or post-remission chronic or accelerated phase, these cases were identified as CML-BP.Results: 31 patients (pts) were identified: 7 (23%) acute biphenotypic leukemia, 11 (35%) CML-BP, 9 (29%) Ph+ AML, and 4 (13%) indeterminate. Among the 9 pts with Ph+ AML the median age at diagnosis was 50 years (range, 35–76), initial white blood cell count 50 (range, 4–210), peripheral blast 64% (range, 0–96), and bone marrow blast 78% (range, 34–98). 7/9 (78%) pts had additional cytogenetic abnormalities apart from t(9;22)(q34;q11), most commonly deletion 7 (n=3). A variety of induction chemotherapy regimens were employed, including cytarabine based (n=6), gemtuzumab ozogamicin-based (n=2), anthracycline based (n=1), and with the addition of imatinib (n=1). 5/9 (56%) pts classified as Ph+ AML achieved a CR or CRp, 3 (33%) died during induction, and 1 (11%) was refractory. 3/5 pts that achieved remission relapsed, with a median CR duration of 35 weeks (range, 20–42) and median OS 58 weeks (range, 55–61). Stem cell transplant was employed in 2 pts, one refractory to induction therapy who died 34 weeks later and one transplanted in CR and alive at 56 weeks; this is also the only pt to be treated with imatinib, as part of induction therapy. The median overall survival for pts classified as Ph+ AML was 34 weeks (range, 2–64) and similar to that of pts reclassified as CML-BP, which was 64 weeks (range, 11–271) (p=0.15).Conclusion: Ph+ AML represents a rare entity that may be mistaken for de novo CML-BP. Herein, we propose a system to help with this differentiation. Clinical outcome is poor with conventional chemotherapy and combinations based on tyrosine kinase inhibitor-based therapy should be investigated.

Irinotecan, Carboplatin, and Imatinib in Untreated Extensive-Stage Small-Cell Lung Cancer: A Phase II Trial of the Minnie Pearl Cancer Research Network

The tyrosine kinase KIT has variable expression in small-cell lung cancer (SCLC) and may be a prognostic factor. Imatinib targets KIT expression, providing rationale for studying its role in combination with chemotherapy in SCLC in a multicenter phase II trial. Patients with untreated extensive-stage SCLC received carboplatin area under the concentration-time curve of 4 on day 1; irinotecan 60 mg/m2 on days 1, 8, and 15; and imatinib 600 mg/day. Treatment cycles were 28 days. Patients remained on imatinib until progressive disease or significant toxicity. Between September 2002 and May 2004, 68 patients were enrolled in this multicenter trial. Median age was 60 years (range, 37-81). The objective response rate was 66% (95% confidence interval: 54%-76%). Median progression-free survival was 5.4 months (95% CI: 4.3-6.0 months). Median overall survival was 8.4 months (95% CI: 6.3-10.5 months). Thirty-five percent of patients were alive at 1 year. Grade 3/4 hematologic toxicity included neutropenia (43%), anemia (16%), and thrombocytopenia (9%). Grade 3 nonhematologic toxicity included diarrhea (19%), fatigue (24%), and nausea (26%). Forty-eight of 56 patients (86%) with available tumor specimens had KIT expression detected. KIT expression did not appear to correlate with progression-free survival or overall survival in a retrospective analysis. Irinotecan, carboplatin, and imatinib is a safe and generally well-tolerated regimen in patients with SCLC. However, the addition of imatinib did not improve results from those expected with chemotherapy alone.

Emerging therapeutic options for Philadelphia-positive acute lymphocytic leukemia

Acute lymphocytic leukemia (ALL) is a heterogeneous group of disorders that are associated with a cure rate of > 80% in children. The prognosis in adults is considerably inferior, with age, disease bulk, leukemia karyotype and immune phenotype being prognostically relevant. Adult ALL treatment programs include induction, intensified consolidation and maintenance phases with CNS prophylaxis. The addition of imatinib in patients with BCR-ABL-positive ALL has improved the prognosis of this subgroup, but their survival is still poor. Initial data on the second-generation BCR-ABL inhibitors, dasatinib and nilotinib, indicate a potentially greater efficacy than imatinib, but the improvement is likely to be modest. The overall efforts in terms of developmental therapeutics in ALL are very modest and not in keeping with the urgent need for improvement. Most agents being investigated have mechanisms of action similar to those of existing agents for ALL therapy and thus represent modest opportunities to improve results. Of such agents, data on BCR-ABL inhibitors, sphingosomal vincristine, pemetrexed, talotrexin, annamycin and ABT-751 are reviewed.

Heat Shock Protein 70 over Expression Is Associated to Imatinib Resistance in Chronic Myelogenous Leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood , 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Hematopoietic Stem Cells Actively Extrude Imatinib-Mesylate Due to High ABCG2-Transporter Activity.

The tyrosine kinase inhibitor imatinib mesylate (STI571) is a potent therapeutic agent for treatment of chronic myeloid leukemia (CML) due to its specific inhibition of bcr-abl kinase. However, patients with CML always relapse after withdrawal of imatinib therapy. With CML being the paradigm for a stem cell disease, we sought to investigate the influence of imatinib on the most primitive stem cell compartment.Imatinib has recently been reported to be a high affinity substrate for the ABC-transporters ABCG2/BCRP (breast cancer resistance protein) and MDR1 (P-Glycoprotein). Given the high expression of these ABC-transporters in Hematopoietic Stem Cells (HSC), we examined the influence of imatinib on ABCG2- and MDR1-activity in human and murine HSC by using Hoechst- and Rhodamine-efflux assays. MDR1-mediated Rhodamine-efflux was only mildly influenced by Imatinib. However, addition of imatinib at therapeutic dosages completely abrogated the SP phenotype of total bone marrow after Hoechst 33342 staining. This effect was even more pronounced in cells of the HSC-phenotype, i.e., lineage-negative/AC133+ or c-kit+/Sca-1+/lin− (KSL) of human and murine marrow, respectively. In order to determine the effect of imatinib on stem cells in vivo, we isolated SP cells from 13 CML patients at various stages of disease using FACS. We employed quantitative RT-PCR-analysis for bcr-abl and demonstrated that the majority of CML patients had bcr-abl-negative SP cells, while peripheral blood mononuclear cells were mostly bcr-abl-positive. However, two patients in complete cytogenetic and molecular-genetic remission proved to be bcr-abl-positive within the SP cell population. This supports the notion that continuous imatinib-therapy may not eradicate the malignant stem cell pool and the leukemic clone may be able to expand even during permanent imatinib-therapy.Furthermore, these data imply that the detoxifying function of ABC transporters on stem cells may be altered under imatinib treatment. Using HEK-293 cells transfected with ABCG2, we demonstrate that imatinib reverses mitoxantrone-resistance - potentially due to high affinity substrate inhibition.In summary, our data suggest the novel resistance mechanism of imatinib being actively extruded from primitive leukemic stem cells, and that addition of chemotherapeutic agents to imatinib therapy may facilitate the eradication of bcr-abl-positive stem cells in chronic myeloid leukemia.

The Impact of Imatinib Therapy on Adult Philadelphia Positive Acute Lymphoblastic Leukaemia (ALL): Early Results from the UKALL12/ECOG 2993 Study.

Modern protocols for the treatment of adults with Philadelphia positive (Ph+ve) ALL produce high complete remission (CR) rates. Despite this, the overall prognosis remains poor. Imatinib, an inhibitor of the BCR-ABL protein tyrosine kinase, may be a useful therapeutic agent for this disease. The arm of the MRC UKALL-XII/ECOG E2993 trial for patients with Ph+ve adult ALL was amended in 2003 to include Imatinib in the treatment protocol. Following induction chemotherapy those in haematological CR went on to receive treatment with Imatinib at 400mg/day, escalated to 600mg/day if tolerated. Those patients with a sibling, matched unrelated donor or related single haplotype match went on to allogeneic transplantation, whilst those with no available donor received an autograft. Chemotherapy alone was given to patients ineligible for a transplant procedure. All patients received Imatinib as maintenance therapy until frank relapse. 30 consecutive patients had a minimum of 12 months follow up. Sufficient data was available in 27 patients. The median age was 45 years (range 15 to 60), 59% were male, 96% were B-lineage, 4% were null lineage. Median white cell count at diagnosis was 24.1 X109/l. Median follow up was 15.6 months. The overall remission rate was 89%. The median time to CR was 28 days (range 19–93 days). Five (19%) patients received an autograft, 15 (56%) received an allograft. and 7 patients (26%) received chemotherapy alone. The event free survival (EFS) and overall survival (OS) at 1 year was 56% and 59% respectively. There were 7 deaths in remission (23%), 6 of which were transplant related. There were 7 relapses, 3 occurred in patients treated with chemotherapy alone, 2 prior to transplantation and 2 following allogeneic transplantation. These results were compared to 236 historic study patients in first CR after the same induction therapy but who did not receive Imatinib. There were no significant differences between the 2 groups with respect to gender, age, WBC or immunophenotype. Four percent received an autograft, 42% received an allograft and 47% received chemotherapy alone. A further 7% received a non-protocol transplant regimen. The EFS and OS of the historic controls was 35% and 56% respectively, with a comparable rate of deaths in remission (22%). Although the superior EFS in the Imatinib treated patients may be confounded by the greater number of allogeneic transplants in that group, the addition of Imatinib, even after achievement of CR does appear to enhance EFS in comparison to that seen in historical controls. Further follow up is needed to confirm an effect on overall survival and an independent anti-leukaemic effect of Imatinib in adult ALL.