Myeloid Blast Crisis Research Articles

Philadelphia Chromosome-Positive Acute Myeloid Leukemia: A Rare Aggressive Leukemia With Clinicopathologic Features Distinct From Chronic Myeloid Leukemia in Myeloid Blast Crisis

Philadelphia Chromosome-Positive Acute Myeloid Leukemia: A Rare Aggressive Leukemia With Clinicopathologic Features Distinct From Chronic Myeloid Leukemia in Myeloid Blast Crisis

Philadelphia Chromosome–Positive Acute Myeloid Leukemia

We performed a multi-institutional retrospective analysis of the morphologic features, immunophenotype, cytogenetics, and BCR-ABL transcript characterization of cases of Philadelphia chromosome-positive acute myeloid leukemia (Ph+ AML). We compared these cases with cases of documented chronic myelogenous leukemia in myeloid blast crisis (CML-MBC). Patients with Ph+ AML were less likely to have splenomegaly or peripheral basophilia and had lower bone marrow cellularity and myeloid/erythroid ratios than patients with CML-MBC. Additional specific cytogenetic abnormalities that typically occur in CML-MBC were less common in Ph+ AML. Of 7 patients with Ph+ AML treated with imatinib mesylate, 6 showed at least a partial hematologic response, but the responses were of a short duration (median, 2.5 months). The median survival of patients with Ph+ AML was 9 months, similar to that of patients with CML-MBC (7 months). Ph+ AML is a rare aggressive acute leukemia with some features distinct from CML-MBC.

Mouse embryonic stem cells that express a NUP98–HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL

NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia and chronic myeloid leukemia blast crisis. We generated 'knock-in' mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to hematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature hematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of erythropoietin (Epo), granulocyte colony-stimulating factor or macrophage colony-stimulating factor. The cell lines are incompletely transformed, as evidenced by their dependence on interleukin 3 (IL-3), and their failure to initiate tumors when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL-3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs hematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation.

Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis

Despite advances in drug therapy and allogeneic stem cell transplantation (allo-SCT), the prognosis of patients with chronic myeloid leukemia (CML) in blast crisis remains poor. Imatinib has demonstrated synergistic effects in vitro with mitoxantrone, etoposide, and cytarabine. A Phase I/II trial was performed in patients with CML myeloid blast crisis. Patients were treated with imatinib + mitoxantrone/etoposide in four cohorts: mitoxantrone 10 mg/m(2)/day and etoposide 100 mg/m(2)/day for 2 or 3 consecutive days and imatinib 600 mg/day from Day 15 (cohorts 1 and 2) or from Day 1 (cohorts 3 and 4). After hematologic reconstitution after the cytopenic phase, cytarabine was given at a dose of 10 mg/m(2)/day in addition to imatinib as maintenance treatment. A total of 16 patients were available for analysis, median age 59 years (range, 37-74). All patients who received more intensive induction treatment (cohorts 3 and 4, n = 7) achieved a hematologic response (HR). In contrast, HR was achieved in only 6 of 9 patients treated in cohorts 1 and 2. The induction treatment was well tolerated. Six patients who achieved HR received an allo-SCT with myeloablative conditioning. The median survival in the transplant group was 16.2 months vs 4.7 months in the group with conventional treatment only (P = .067). The combination of mitoxantrone/etoposide and imatinib is well tolerated, with mild nonhematologic toxicity even in older patients. Eligible patients benefit from allo-SCT after response to the induction treatment.

Chronic Myeloid Leukemia Blast Crisis Arises from Progenitors

Chronic myeloid leukemia (CML) progresses through three distinct clinical stages: chronic phase, accelerated phase, and blast crisis. The progression to accelerated phase and blast crisis is driven by activation of oncogenes, inactivation of tumor suppressor genes, and/or amplification of the BCR-ABL fusion gene, which causes the chronic phase of the disease. The cell of origin of blast crisis is a subject of speculation. Here, I develop a simple mathematical model of CML blast crisis to investigate whether blasts arise from leukemic stem cells or more differentiated leukemic cells. I use data of patients treated with imatinib and previous agents to estimate the effects of therapy on the rate of progression. Imatinib reduces the progression rate 10-fold as compared with previous (ineffective) therapies. If blasts were produced by leukemic stem cells, there would be no difference in the rate of progression between patients treated with imatinib and previous therapies, because imatinib seems to be incapable of depleting leukemic stem cells. Imatinib does, however, deplete leukemic progenitors. Therefore, CML blasts are likely to arise from leukemic progenitors. Disclosure of potential conflicts of interest is found at the end of this article.

Contribution of ABL Kinase Domain Mutations to Imatinib Resistance in Different Subsets of Philadelphia-Positive Patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia

ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Response to Dasatinib after Imatinib Failure According to Type of Preexisting BCR-ABL Mutations.

Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases with preclinical activity against 20/21 imatinib resistant BCR-ABL mutations and clinical phase I/II efficacy in patients with chronic myelogenous leukemia (CML) and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and efficacy of dasatinib in patients (pts) with CML and ALL. We have investigated 872 peripheral blood samples from 394 pts (53% male, median age 60 yrs, range 17–85) who had been enrolled in international phase II studies investigating the activity of 70mg dasatinib BID after imatinib failure (chronic phase, CP, n=198; accelerated phase, AP, n=78; myeloid blast crisis, MyBC, n=53; lymphoid blast crisis, LyBC, or ALL, n=65).Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Hematologic and cytogenetic response data have been collected sequentially for a median of 8 months (range, 2–11) after start of therapy. Prior to dasatinib, 46 different BCR-ABL mutations involving 36 amino acids were detected in 202/394 pts (51%). 162 pts showed one, 33 pts two, 6 pts three, and 1 pt four mutations. Mutations were observed in 84 pts in CP (42%), 47 pts in AP (60%), 23 pts in MyBC (43%), and 48 pts in LyBC and ALL (74%). In patients with mutations, hematologic response was 91% in CP, 62% in AP, 41% in MyBC, and 34% in LyBC/ALL (p<0.01), major and complete cytogenetic response did not differ significantly (47% and 34% in CP, 35% and 27% in AP, 33% and 28% in MyBC, 53% and 51% in LyBC/ALL). Major cytogenetic response rates were comparable in pts bearing no mutations (44%), mutations within the P-Loop (43%), SH2 domain (47%), activation loop (56%), or other sites (49%), but not for T315I (0%, p<0.001). Sorting individual pts by the underlying mutation and cellular IC50 values of dasatinib revealed clearly higher hematologic and cytogenetic response rates in those with lower IC50 values. In line with the virtual insensitivity to dasatinib in vitro, none of the 17 pts carrying a T315I mutation showed any hematologic response. Two distinct patterns of response were observed:a parallel decrease of the BCR-ABL load and the mutated clone ora decrease of the BCR-ABL load followed by a decrease of the mutated clone after a delay of up to 4–6 months.Up until now 5 pts developed new mutations associated with dasatinib resistance (T315I, n=2, AP and MyBC pts; F317L+F486S, n=2, LyBC and MyBC pts; E507G, n=1, CP pt). We conclude that dasatinib is capable of inducing hematologic and cytogenetic remissions in a significant proportion of imatinib resistant pts harboring BCR-ABL mutations, except T315I. Response dynamics depend on the individual type of mutation which may be a basis for individual dose adaptation according to the mutation pattern.

Analysis of GATA-1 Mutations in Chronic Myeloid Leukaemia.

Despite extensive characterisation of the molecular pathogeneisis of chronic myeloid leukaemia (CML), little is known about the events underlying progression to accelerated phase and blast crisis. The haematopoietic transcription factor GATA1 plays an essential role in the commitment to, and differentiation within, erythroid, megakaryocyte, eosinophil and mast cell lineages. The observation of frequent eosinophilia and thrombocytosis alongside granulocytic hyperplasia suggests that GATA1 is actively expressed in CML chronic phase. GATA1 is mutated in the acute megakaryoblastic leukaemia of Down syndrome and in its preleukaemic form, transient abnormal myelopoiesis. The latter can morphologically resemble CML with basophilia and eosinophilia in addition to circulating blast cells.Examination of the expressed sequence tag (EST) database for GATA1 shows 4 variant sequences. 3 of these are derived from CML patient bone marrow.Given the plausible association between perturbations in GATA1 function and progression of CML, along with suggestive EST evidence, we hypothesized that mutations in GATA1 could be involved in the transition from chronic phase to blast crisis in CML.Materials and methods: Following appropriate informed consent and ethical approval genomic DNA was extracted from blast cells from 24 patients with CML in accelerated phase (n=1), myeloid blast crisis (n=15) or lymphoid blast crisis (n=8). Polymerase chain reaction (PCR) products were generated encompassing each coding exon of GATA1 and about 50 base pairs of flanking sequence. Exons 3 and 6 were divided into 2 fragments each for analysis. Gel purified PCR products were screened for mutations in GATA1 using DHPLC (denaturing high pressure liquid chromatography, WAVE, Transgenomics, USA). Samples with abnormal migration profiles were directly sequenced by bi-directional sequencing using an ABI 3100 Capillary Array sequencer (Perkin Elmer, UK). Mutations detected by this method were confirmed by a repeat PCR reaction, Topo cloning into pCR4TOPO vector (Invitrogen) and commercial sequencing (Agowa, Germany)Results and discussion 2/24 samples showed abnormal migration patterns by WAVE analysis. Sequencing confirmed 2 different single nucleotide changes in the intronic sequence flanking exon 5. These do not correspond to any known single nucleotide polymorphisms. Since these 2 mutations do not arise in coding or known regulatory regions of the gene it seems unlikely that they are of functional significance. Abnormal regulation of GATA1 activity either at the transcriptional or post-translational level may still play an important part in the pathogenesis of the disease. The mitogen-activated protein kinase (MAPK) pathway has recently been shown to be important in controlling the oncogenic potential of bcr-abl. MAPK mediated phosphorylation of GATA1 at critical serine residues promotes cell survival via increased Bcl-XL expression. In addition study of the upregulation of inducible heat shock protein 70 (Hsp70) in CML has shown the major p210 bcr-abl responsive element upstream of the Hsp70 promoter to be a GATA binding motif.Conclusion These findings suggest that GATA1 mutations are not frequent occurrences in the evolution of blast crisis from CML chronic phase. However, this study does not rule out an important role for dysregulated GATA1 activity in the molecular pathogenesis of chronic myeloid leukaemia.

GM-CSF Secreting Leukemia Cell Vaccinations after Allogeneic Reduced-Intensity Peripheral Blood Stem Cell Transplantation (SCT) for Advanced Myelodysplastic Syndrome (MDS) or Refractory Acute Myeloid Leukemia (AML).

Disease relapse is a frequent cause of treatment failure in patients undergoing reduced intensity allogeneic stem cell transplantation for advanced MDS and AML. Prior studies with GVAX, a cancer vaccine composed of irradiated autologous myeloblasts modified to secrete GM-CSF, suggested anticancer activity in MDS and AML after autologous SCT or in primary therapy. We investigated the feasibility and safety of administering GVAX after allogeneic SCT. Patients with MDS-RAEB or AML with >5% marrow blasts and with a donor matched at HLA-A,B, DRB1 were eligible. Prior to transplant, autologous myeloblasts were collected from the marrow or blood and transfected with an adenovirus vector bearing the GM-CSF gene to generate the GVAX vaccine. Patients were conditioned with fludarabine 30mg/m2/d IV days -6 to -3, and busulfan 0.8mg/kg IV q12H days -6 to -3 prior to allogeneic PBSC infusion. GVHD prophylaxis included tacrolimus and mini-methotrexate. GM-CSF (Leukine) 250 mg/m2 SC QD was administered from day +1 until neutrophil engraftment. GVAX was administered SC/ID weekly for the first three doses, then q2 weeks for the last three doses starting between day +30 to +45 if there was neutrophil recovery and no grade II-IV acute GVHD. Taper of tacrolimus began after vaccine completion. Eighteen patients (10 URD, 8 MRD) have been transplanted to date: 12 AML, 4 MDS/RAEB, 2 CML myeloid blast crisis. Median age was 63 (range, 41–71 years). Median marrow blast content at transplant was 31% (range, 6–91%). GVAX was successfully generated for all 18 patients. Median vaccine cell dose was 1.0 × 107 cells (range, 0.1–1.0 ×107), and median 24-hour GM-CSF secretion by the vaccine was 8.2 ng/ml/106 cells (range 0.4 – 195). Seven patients did not initiate vaccination due to: inadequate neutrophil recovery/leukemia progression (4); acute GVHD (2); sepsis/death (1). Two patients were recently transplanted and have not reached day 30 to start vaccination. Among 9 patients who received GVAX, vaccination was well tolerated. Mild injection site reaction with induration, erythema, or pruritus was the only common side effect. After vaccination, 1 patient developed grade II skin acute GVHD and 3 had cGVHD that resolved with steroids. Donor chimerism was not adversely affected by vaccination. Six of 9 patients who started GVAX are alive at a median follow up of 7.5 months post transplant (range 1–16 mos). These include 4 patients in complete remission (3 AML, 1 MDS-RAEB) between 5 and 16 months after transplant, and 2 patients with minimal persistent AML early after SCT. Four of 5 patients who completed 6 vaccines remain progression free. Histologic examination of vaccination and leukemia cell DTH sites revealed significant infiltration with inflammatory cells and eosinophils. These preliminary results suggest GVAX vaccination is safe and may have anticancer activity in patients with MDS/AML after reduced intensity allogeneic SCT.

Prolonged Engraftment and/or Primary Graft Failure Following Allogenic Stem Cell Transplant in Patients Treated with Dasatinib.

The impact of pre-transplant dasatinib therapy on engraftment after allogenic stem cell transplant remains unknown. We report delayed engraftment in the first three consecutive chronic myeloid leukemia (CML) patients at our center who received allogenic stem cell transplant after treatment with dasatinib for imatinib mesylate resistant (CML). All the patients were enrolled on the BMS clinical trial CA180035 for accelerated or blast phase chronic myeloid leukemia resistant or intolerant to imatinib. All three patients received G-CSF 5mg/kg sc daily starting from day +1 × 3 days post transplant to achieve ANC >1000. Patient 1: 53 yr female developed lymphoid blast crisis on imatinib and was given one cycle of HyperCVAD, entered a complete molecular remission (RT-PCR/BCR-ABL fish negative) and then received 140 mg PO daily of dasatinib continuously for 100 days prior to transplant. Dasatinib was discontinued one week prior to transplant and the patient was conditioned with fractionated TBI/CY 60mg/kg × 2 and infused with 2.32× 106 CD34/kg BM from a 10/10 matched unrelated donor. Patient 2: 36 yr male with accelerated phase (CML) failed imatinib and developed myeloid blast crisis. After starting dasatinib 70mg PO BID patient remained in accelerated phase and was taken off study drug after 82 days approximately two months prior to transplant because of recurrent blast crisis requiring admission for standard anti-leukemic therapy. This patient was conditioned with BU/CY and infused with 16.2× 106 CD 34/kg PBSC from a 9/10 (C antigen mismatched) unrelated donor. Primary graft failure with recurrence of disease was documented on day 30 requiring additional investigational therapy. Patient 3: 61 yr female with accelerated phase (CML) was treated with dasatinib 70mg PO BID after progression on imatinib and was unable to continue on dasatinib after 20 weeks due to myleosuppression. Dasatinib was discontinued one month prior to transplant and no additional therapy was needed. She received single dose 550 cGy TBI/CY 60mg/kg × 2 followed by infusion of 6.9 106 CD34/kg PBSC from a HLA-matched sibling donor. Primary graft failure occurred and patient was re-infused with additional G-mobilized PBSC (5.5 × 106 CD34/kg) on day +30. Patient did not achieve neutrophil recovery until 5 days after second infusion of G-mobilized PBSC from the same donor. Although the number of patients is limited, 3/3 patients treated with dasatinib prior to allogenic transplant experienced either delayed engraftment or primary graft failure. Additional patients treated with dasatinib prior to allogenic transplant will need to be studied to confirm these observations and to provide more information on dose and duration of dasatinib therapy that may impact hematopoietic engraftment after myeloablative allogenic stem cell transplantation.Patient CharacteristicsPatients123Type of TransplantBMPBPBCD34/kg2.32×10^6/kg16.2×10^6/kg6.9×10^6/kgConditioningTBI/CYBU/CYTBI/CYDisease status at transplantCMRAP CMLAP CMLANC 500+21+27+30ANC 1000+25+27+38PLTS 25+21Not AchievedNot AchievedPLTS 50+25Not AchievedNot AchievedDonor Chimerism100% day +300% day +3073% day+24/100% day+84

Sequential Use of Recombinant Factor VIIa and FEIBA in a Pediatric Patient with Disseminated Intravascular Coagulation.

The persistence of abnormal coagulation test results and bleeding after standard treatment in the setting of disseminated intravascular coagulation (DIC) can pose a significant challenge. Recombinant Factor VIIa (Novo Nordisk) has been used in the pediatric population for treatment of hemophilia patients with inhibitors and in various other instances as a single agent to manage coagulopathy. (1) The use of NovoSeven and FEIBA (Baxter Hyland Immune) has been reported in use of adult patients with factor deficiencies and coagulation factor inhibitors. (2) The synergistic use of NovoSeven and FEIBA to improve clinical bleeding caused by DIC in a pediatric patient is being described.A 15 year old African American male presented with a 2 month history of leg pain and a WBC of 400K. He was diagnosed with Philadelphia chromosome positive CML and treated with Allopurinol and Imatinib. After 7 months on Imatinib, he presented in septic shock. His WBC increased to 50.8K with 44% myeloblasts. Flow cytometry confirmed myeloid blast crisis. The patient was admitted to the PICU on broad spectrum antibiotics. Within 12 hours of admission, he required ventilatory assistance. Central lines were placed in the right subclavian vein, right femoral vein and left radial artery. He began bleeding from his nostrils, endotracheal tube and all of his central line sites. Mitoxantrone and Cytarabine were started. Patient was noted to have DIC with PT> 20 sec, APTT> 40 sec, platelets of 23K and D-Dimers of 11.86 (normal <0.42 mcg/ml). His Fibrinogen level was maintained above 300mg/dl during the first 8 days of admission. By hospital day 2, the patient received numerous units of FFP, platelets and PRBC, but he continued to bleed. NovoSeven was started due to volume concerns and increased bleeding. He had improvement in his coagulation tests but no improvement in his bleeding. On hospital day 3, NovoSeven was given intravenously alternating with FEIBA. The patient received NovoSeven 100 micrograms/kg IV q 12 hours. About 6 hours from the NovoSeven dose, FEIBA was given on a q 12 hour schedule at 50 units/kg IV. Significant improvement was seen with the coagulation profile immediately and the bleeding improved in 6 hours. The patient was discharged from the PICU 15 days from his admission with no clinical adverse events associated with the administration of the NovoSeven and FEIBA. He has subsequently tolerated a haploidentical transplant from his sister and he remains in continuous remission.Nigel Key and colleagues have described improved clinical clotting response with prothrombin complex concentrate (PCC) and NovoSeven. They hypothesized that the coagulation proteins in PCC have a longer half life impacting on the effectiveness of NovoSeven. (2) The use of FEIBA and NovoSeven is risky without a widely available clinical assay to assess responsiveness to NovoSeven. This anecdotal report suggests that FEIBA and NovoSeven can be given sequentially without adverse thrombotic events. Further study in monitoring NovoSeven and using sequential agents may be helpful in patients who prove unresponsive to NovoSeven alone.

A Ph-Negative Myeloproliferative Disorder with JAK2 Mutation Disclosed by Imatinib Therapy in Two Patients with Ph+ CML.

Protein tyrosine kinases play a crucial role in normal and neoplastic cell development. Recently, a unique clonal mutation in the JAK2 gene that results in a valine to phenylalanine substitution at position 617 (V617F), leading to constitutive JAK2 activation and abnormal signaling, was found in the majority of BCR-ABL negative chronic myeloproliferative disorders (CMPD) patients. Although JAK2 activation by tyrosine phosphorilation is involved in the BCR-ABL transformation of CML cells, V617F mutation is not usually observed in Ph-positive leukemias.We describe two patients with BCR/ABL+ (p190) CML which followed a Ph- CMPD characterized by JAK2 mutation.Patient 1 was diagnosed as having a idiopathic myelofibrosis in Oct 1997. At that time a Ph-chromosome was not detected. The patient remained untreated until March 1999, when WBC started to increase up to 90.000/cmm with a parallel drop in platelet count and increase in spleen size. Hydroxiurea was started with partial hematological remission. In Jan 2000 the presence of a BCR/ABL transcript (p190 type) and a Ph-chromosome in 84% of the cells were observed. Imatinib treatment resulted in a initial improvement, soon followed by progressive increase in the WBC and platelet counts, in spite of a reduction of Ph+ cells to 4%. Hydroxyurea was added but hematological control remained unsatisfactory and imatinib treatment was stopped after 6 months. The patient continued on hydroxyurea for the following year. In 2002 he developed a myeloid blast crisis; karyotype was normal, 4.8% of the cells were Ph+ at FISH and the p190 transcript was still present at low levels.Patient 2 was diagnosed as essential throbocythemia in 1990; cytogenetic analysis showed a normal karyotype. A good control of the thrombocytosis was obtained with hydroxyurea for more than 10 years. In 2003 the hematological data showed leukocytosis (up to WBC 42.900/cmm), immature myeloid cells and 16% monocytes in PB. A masked Ph chromosome was observed by FISH in 47% of cells. RT-PCR was positive for a p190 transcript. The patient started therapy with imatinib which induced a rapid decrease in WBC count; however, the platelet count rised up to 977.000/cmm and hydroxiurea was added again. In subsequent bone marrow samples a progressive decrease of BCR/ABL positive cells to 2% was observed and a BCR-ABL/ABL ratio of 0.18 % was reached in January 2006. However, continuous hydroxyurea therapy was needed to partially control thrombocytosis.Both patients were tested for the presence of JAK2 mutation using the ABI Prims 3100 genetic analyzer and a JAK2-V617F mutation was present. Presence of two clones, possibly derived from a common abnormal stem cell, can be hypothesized in these cases. The possibility of a multistep patogenesis for CML has been widely discussed; in these patients, CML apparently developed as a late event, partially replacing a Ph-CMPD cell population characterized by the JAK2 mutation. Imatinib treatment allowed the reexpansion of the Ph- cells resulting in a overall poor hematological control. Both patients only displayed p190 BCR/ABL transcript, a quite unusual finding in chronic phase CML. It is not clear if any relationship may sussist between this molecular event and the unusual clinical history of these patients.

BCR-ABL Activates IGF-1 Expression and Signaling in Chronic Myelogenous Leukemia Blast Crisis Cell Lines.

CML blast crisis is characterized by the continued presence of the Philadelphia chromosome, which expresses the P210 BCR-ABL fusion protein, and the acquisition of additional molecular and chromosomal alterations. Evolution from CML chronic phase to blast crisis is associated with loss of heterozygosity at chromosome region 1p36, which contains the putative tumor suppressor RIZ1. We found that RIZ1 expression decreases during progression from CML chronic phase to myeloid blast crisis and that forced RIZ1 expression in CML blast crisis (CML-BC) cell lines decreases proliferation, increases apoptosis, and enhances differentiation. Furthermore, we found that RIZ1 represses IGF-1 autocrine production and blocks the activation of the IGF-1 receptor, AKT, and ERK signaling pathways in CML-BC cell lines thereby implicating RIZ1 control of the IGF-1 pathway in the regulation of these phenotypes. As BCR-ABL induces a growth factor independent phenotype due to the activation of autocrine growth factor production, we analyzed IGF-1 expression in CML-BC cell lines following exposure to imatinib. We found that imatinib treatment reduced IGF-1 mRNA and extracellular IGF-1 suggesting that RIZ1 may suppress blast crisis by counteracting the ability of BCR-ABL activate RIZ1 regulated genes. We characterized the signaling pathways used by BCR-ABL to activate IGF-1 expression and found that the HCK inhibitor PP2 and STAT5b shRNA reduced IGF-1 expression whereas the JAK2 inhibitor AG490 increased IGF-1 expression. These results suggest that BCR-ABL activates HCK, which in turn activates STAT5b and induces IGF-1 expression. To confirm the importance of BCR-ABL induced IGF-1 signaling to CML-BC cell line viability, proliferation and apoptosis, we characterized these cellular phenotypes in the presence of IGF-1 receptor blocking antibody and the IGF-1 receptor tyrosine kinase inhibitor AG1024. Blockage of autocrine IGF-1 signaling in CML-BC cell lines using an anti-IGF-1 receptor blocking antibody reduced cell viability and decreased proliferation. AG1024 treatment of CML-BC cell lines decreased proliferation and induced apoptosis. Together these results demonstrate that RIZ1 counteracts the ability of BCR-ABL to induce IGF-1 signaling in CML-BC cell lines, which influences cell proliferation, apoptosis, and differentiation. Our findings highlight RIZ-1 and IGF-1 signaling pathways as potential therapeutic targets for treating CML blast crisis.

Phase I Study of Cladribine, Cytarabine (Ara-C), Granulocyte Colony Stimulating Factor (G-CSF) (CLAG Regimen) and Simultaneous Escalating Doses of Imatinib Mesylate (Gleevec) in Relapsed/Refractory AML.

Background: Receptor activated tyrosine kinases like c-kit, c-fms and PDGFR are known targets of inhibition by imatinib mesylate (Gleevec) and are expressed on AML blasts. Marrow stromal cells and monocytes express the ligands KIT ligand, M-CSF and PDGF capable of activating survival pathways in these leukemic cells. Imatinib mesylate is a substrate of the multidrug resistance and ABC - transporters expressed in AML cells and displays a modest activity alone in AML in vitro and in vivo. Given the synergy in vitro between Ara-C and Gleevec on AML cell growth inhibtion, we initiated a Phase I study combining CLAG + Gleevec in AML patients.Method: Patients with relapsed, refractory AML or CML myeloid blast crisis were eligible to receive Cladribine 5 mg/m2 Days 3–7, Cytarabine 2 gm/m2 Days 3–7, G-CSF 300 mcg Days 2–7, and escalating doses of imatinib mesylate given on Days 1–15. The level 1 Gleevec dose was 400 mg, while level 2 was 600 mg and the level 3 dose 800 mg. Patients were allowed to receive a second course of treatment if a CR was not attained after the first course, and were eligible for allogeneic stem cell transplantation (ASCT) if a donor was available.Results: A total of 15 patients have been enrolled 14 AML and 1 CML blast crisis, 6 patients with relapse within 6 months following initial induction/consolidation, 5 patients relapsed within 6–12 months of therapy, 3 patients had refractory disease and 3 patients relapse following myeloablative ASCT. No dose limiting toxicity was reached. The dose escalation occured as planned and there was no clear evidence of added toxicity due to Gleevec. One patient with an extensive cardiac history died of cardiac causes on day 1 of therapy, no other deaths occured within 30 days of starting therapy. One patient had a grade 3 skin rash at level 2 and did not receive imatinib on the second induction therapy. None of the 15 patients treated have had a drug- related cause of death: the most common toxicities encountered during induction therapy were nausea, vomiting, ALT and AST increase, rash and diarrhea and were transient and/or reversible. There were two drug-nonrelated deaths one CNS bleed in a persistently thrombocytopenic patient after ANC recovery and ovelwhelming sepsis in another patient. Out of 14 evaluable patients 12 achieved a hypocellular marrow after initial induction with one additional patient achieving a hypocellular marrow following a second course of the same regimen. One patient had persistence of disease and was switched to another treatment. Out of the 12 evaluable patients 5 acheived a complete response (42%) with 4 of these patients achieving a complete cytogenetic response as well (33%). The relapse free interval ranged from 35 to 206 + days in evaluable patients. Imatinib mesylate (up to 800 mg) plus CLAG is a safe and well tolerated non anthracycline based salvage regimen in patients with relapsed/refractory AML.

RIZ1 Is Downregulated during CML Progression and Displays Tumor Suppressor Properties in CML Cell Lines.

Transition to CML blast crisis is characterized by the continued presence of the Philadelphia chromosome and the acquisition of additional molecular and chromosomal alterations. Loss of heterozygosity at chromosome region 1p36 is a frequent finding in CML blast crisis. RIZ1 is located at 1p36 and frequently undergoes deletion, rearrangements, and loss of heterozygosity in a variety of cancers. Taken together, these data suggest that decreased RIZ1 expression may contribute to CML progression. We used immunohistochemistry to analyze RIZ1 expression in matched bone marrow biopsy specimens from CML chronic phase patients that progressed to accelerated phase or myeloid blast crisis. Similar to control bone marrow, strong cytoplasmic and nuclear RIZ1 expression was observed during chronic phase in all cases. However, RIZ1 expression was found to be markedly decreased in matched bone marrow biopsy specimens obtained during myeloid blast crisis in five patients. RIZ1 expression was maintained in the immature cells of two CML patients, one in accelerated phase with 15% myeloid blasts and the other in myeloid blast crisis, indicating that low RIZ1 expression is not an inherent property of immature hematopoietic cells. To confirm this, we analyzed G-CSF mobilized peripheral blood by flow cytometry and found RIZ1 to be expressed in mature myeloid and CD34+ cells. Transient transfection of myeloid blast crisis cell lines K562, YN-1, ERY-1, and JURL-MK1 with a RIZ1 expression plasmid (pRIZ1) increased the number of cells undergoing early and late apoptosis and reduced viability by 20–80% within 24 hours. RIZ1 effects on erythroid differentiation were assessed in K562, YN-1, and ERY-1 as these cell lines express low levels of hemoglobin, reflecting their myeloid/erythroid progenitor phenotype. As transient RIZ1 expression in these cells is too toxic to measure erythroid differentiation, we modified K562 to express less toxic levels of RIZ1 by stably integrating RIZ1 under the control of a CMV promoter (K562+RIZ1). As determined by benzidine staining, stable expression of RIZ1 in K562+RIZ1 increased erythroid differentiation compared to K562 alone. To confirm that RIZ1 is responsible for the enhanced erythroid differentiation, we transfected K562+RIZ1 as well as ERY-1 and YN-1 (which have higher endogenous RIZ1 levels than K562), with a plasmid that expresses RIZ1 shRNA (pRIZ1shRNA). Expression of pRIZ1shRNA in K562+RIZ1 reduced RIZ1 protein expression and erythroid differentiation to levels similar to that observed in K562 and decreased erythroid differentiation in ERY-1 and YN-1. RIZ1 effects on JURL-MK1 differentiation were assessed by measuring CD33 and CD117 as the expression of these proteins decreases during myeloid differentiation. Transient transfection of JURL-MK1 with pRIZ1 decreased CD33 and CD117 expression as assessed by flow cytometry. In summary, our study demonstrates that RIZ1 expression is frequently reduced in myeloid blast crisis. Furthermore, RIZ1 expression decreases cell proliferation, increases apoptosis, and enhances differentiation in cell line models of CML myeloid blast crisis. Taken together, our results build upon previous observations that a putative CML tumor suppressor gene is present at 1p36 and suggest that deregulation of RIZ1 expression may contribute to CML progression.

Dynamics of Cytogenetic Aberrations in Philadelphia Chromosome Positive and Negative Hematopoiesis during Dasatinib Therapy of Chronic Myeloid Leukemia Patients after Imatinib Failure.

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of CML to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon alpha or imatinib in a minority of patients (pts) with cytogenetic response. Conflicting data suggest selection of preexisting clones vs. induction of aneuploidy by tyrosine kinase inhibitors. The prognostic impact of aberrations in the Ph negative hematopoiesis for the individual pts remains to be determined. Dasatinib is a multitargeted agent inhibiting both ABL and SRC tyrosine kinases. The efficacy and safety of dasatinib has been demonstrated in phase I and II studies in pts with Ph positive CML after failure of imatinib therapy. We sought to evaluate the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and Ph negative metaphases during dasatinib therapy. 71 pts (40 m, 31 f) with Ph positive CML after failure of imatinib therapy have been evaluated. Median age was 58 years (range, 28–78), median time from diagnosis 73 months (range, 14–231). Pts were in chronic phase (CP, n=50), accelerated phase (AP, n=6), myeloid (n=8) or lymphoid (n=7) blast crisis (BC). Pretreatment consisted of imatinib (n=71), hydroxyurea (n=48), interferon alpha (n=49), cytosine arabinoside (n=11) and nilotinib (n=2). Dasatinib therapy was commenced at a dose of 100–140 mg/day, median duration of dasatinib therapy was 9 months, (range, 1–16). Prior to dasatinib therapy, 22 pts (31%) demonstrated additional chromosomal aberrations in the Ph positive clone indicating BCR-ABL independent mechanisms of resistance. Of these, 8 pts were in CP (16%), 5 in AP (83%), and 9 in BC (60%). In 35 pts (49%), BCR-ABL mutations associated indicating a BCR-ABL dependent mechanism of resistance were observed. 9 pts (13%) showed both clonal evolution and BCR-ABL mutations. Two pts (3%) had trisomy 8 as an aberration of the Ph negative clone at baseline. During dasatinib therapy, 33 pts (46%) achieved major cytogenetic remission, 26 (37%) being complete. From pts with clonal evolution, 3 (14%) achieved a major with 2 (9%) complete cytogenetic remissions. Novel aberrations of the Ph positive clone appeared during dasatinib therapy in 6 pts (8%), aberrations of the Ph negative clone in 3 pts (4%). In total, 5/71 pts (7%) showed clonal aberrations of Ph negative metaphases after consecutive imatinib/dasatinib therapies. None of these pts had morphological indications for secondary neoplastic changes, like myelodysplasia or acute leukemia. In conclusion, dasatinib was efficacious in pts with clonal cytogenetic aberrations as a marker of BCR-ABL independent imatinib resistance. However, cytogenetic response was delayed compared to pts without additional aberrations. Preexisting clonal aberrations of the Ph negative hematopoiesis were uncovered by a simultaneous gradual elimination of the Ph positive clone. In addition, a minority of pts demonstrated novel aberrations in Ph negative cells. The prognostic significance of aneuploidy of the Ph negative hematopoiesis remains to be evaluated by long term observation.

Dual PPARα/γ Ligand TZD18 Either Alone or in Combination with Imatinib Inhibits Proliferation and Induces Apoptosis of Human CML Cell Lines

Despite progress in the treatment of early-stage chronic myeloid leukemia (CML), the accelerated and blastic phases of CML still remain a therapeutic challenge. Persistence of BCR-ABL-positive (bcr-abl+) cells or secondary resistance during imatinib therapy frequently occurs. In this study, we investigated the activity of a novel dual ligand specific for peroxisome proliferator-activated receptor α and γ (PPARα/γ) against CML blast crisis cell lines. Exposure of these cell lines (K562, KU812 and KCL22) to TZD18 resulted in a growth inhibition in a dose- and time-dependent manner. This effect may not be mediated through PPARγ and/or PPARα activation, since antagonists of PPARg and/or PPARa could not reverse this inhibition. Western blotting analysis showed that expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1 was enhanced, whereas levels of cyclin E, cyclin D2 and cyclin dependent kinase 2 (CDK-2) were decreased when these cells were treated with TZD18. Most interestingly, TZD18 synergistically enhanced the antiproliferative and pro-apoptotic effects of imatinib. Overall, our findings strongly suggest that TZD18, either alone or in combination with imatinib may be beneficial for the treatment of CML in myeloid blast crisis.

NF‐kB inhibition as a strategy to enhance etoposide‐induced apoptosis in K562 cell line

NF-kB is a transcription factor that mediates antiapoptotic signals in several cancer cell lines. Here we have demonstrated that the cytotoxic drug, Etoposide, activates NF-kB in K562, a chronic myeloid leukemia blast crisis cell line. Treatment with the NF-kB inhibitors MG-132, Bay11-7082, and Resveratrol impedes Etoposide-induced NF-kB activation, rendering K562 sensitive to Etoposide-induced apoptosis. Stable expression of mutant form of IkB-alpha, which retains NF-kB inactive in the cytoplasm of cells, confirmed the data obtained with molecular inhibitors. Both inhibitors and stable expression of SR-IkB are associated with down-modulation of the antiapoptotic protein Bcl-xL, suggesting that the survival pathway activated by Etoposide involves NF-kB-mediated Bcl-xL expression.

Update on practical aspects of the treatment of chronic myeloid leukemia with imatinib mesylate

Imatinib (imatinib mesylate, Gleevec(R) [formerly known as STI571], Novartis Pharmaceuticals, Basel, Switzerland) is a protein tyrosine kinase inhibitor that is approved by the US Food and Drug Administration for patients with all phases of chronic myeloid leukemia (CML). Imatinib is remarkably effective as treatment for CML in the chronic phase (at a dosage of 400 mg/d) and the accelerated phase (at 600 mg/d). At this time, it remains to be seen whether the chronic phase of CML can be extended sufficiently in some patients so that they are functionally "cured," and also whether the increased rate of major molecular response induced by doses of imatinib higher than 400 mg/d will further improve overall survival of patients with CML in the chronic phase. The value of molecular monitoring of response in patients with CML in the chronic phase is examined. Although imatinib 800 mg/d can induce dramatic responses in patients with myeloid blast crisis, lymphoid blast crisis, and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), the responses are usually incomplete and of short duration. We discuss the role of imatinib in relation to allogeneic stem cell transplantation (particularly in younger patients), recognizing that the data upon which any decisions can be made are relatively immature. Finally, recent data on new tyrosine kinase inhibitors capable of overcoming primary or acquired resistance to imatinib are reviewed.

Dasatinib (D) in patients (pts) with chronic myelogenous leukemia (CML) in myeloid blast crisis (MBC) who are imatinib-resistant (IM-R) or IM-intolerant (IM-I): Results of the CA180006 ‘START-B’ study

6529 Background: Dasatinib (D) (BMS-354825) is an oral, multitargeted tyrosine kinase inhibitor of Bcr-Abl and SRC with activity against IM-R cell lines. In a phase I study, there was preliminary evidence that D was active in MBC-CML patients (pts). Methods: START B is an open-label study of D in IM-R or IM-I MBC carried out in 46 sites worldwide. From December 2004 to May 2005, 124 MBC pts were accrued. D was given orally at 70 mg twice a day (BID) with dose escalation to 100 mg BID for poor initial response or dose reductions to 50 mg and 40 mg BID for toxicity. Pts had weekly blood counts and monthly bone marrow evaluations, including cytogenetics. The primary endpoint was confirmed (minimum 4 weeks duration) major hematologic response (MaHR). Results: Data are currently available on the first 74 pts (68 IM-R, 6 IM-I). Median age was 55 years, 55% were male. Other prior therapy included interferon in 55% of pts and bone marrow transplant (BMT) in 12%. Prior IM dose was &gt;600 mg/day in 49% of pts and 47% of pts received IM for &gt; 3 years. At baseline, the WBC count was ≥20 × 103/mm3 in 46%, the platelet count was &lt; 100 × 103/mm3 in 72% of pts and 35% of pts had ≥ 50% bone marrow blasts. Mutations in the BCR-ABL domain were found in 27/63 (43%) pts. Median duration of therapy was 3.5 months. D doses were reduced in 35% pts, temporarily interrupted in 58% pts, and escalated in 41% pts. With a minimum of 6-month follow-up, hematologic response was seen in 39 (53%) pts: confirmed MaHR in 24 (32%) pts, Complete in 18 (24%) and No Evidence of Leukemia in 6 (8%). Major cytogenetic responses were documented in 22 (30%) pts and was complete in 20 (27%). The median time to MaHR was 56 days. There was no loss of response in pts with MaHR; the duration of MaHR ranged from 1.2+ to 7.8+ months. The median PFS had not been reached. Severe myelosuppression was very common. Non-hematologic toxicities were usually mild to moderate. Most common Grade 3–4 toxicities included diarrhea in (7%), pleural effusion (9%), nausea (4%). Peripheral edema was reported in 14% of pts (0% Grade 3–4), and rash in 11% of the pts (0% Grade 3–4). Conclusions: Dasatinib was highly effective in IM-R pts with MBC with durable MaHR. Data on all 124 pts will be presented at the meeting. [Table: see text]