BCR-ABL Positive Acute Lymphoblastic Leukemia Research Articles

The Analysis of HDAC Inhibitor, Vorinostat Efficacy against Wild Type and BCR-ABL Mutant Positive Leukemia Cells in Monotherapy and in Combination with a Pan-Aurora Kinase Inhibitor, MK-0457

The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.

Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress.In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.

Integrin Alpha M Chain Expression at Diagnosis Is Inversely Correlated with Cytoreduction Rate and Is Consistently Up-Regulated during Therapy in Acute Lymphoblastic Leukemia (ALL)

In childhood ALL, detection and quantification of minimal residual disease (MRD) is of crucial prognostic significance. Gene expression pattern of MRD cells undergoes treatment-related changes which provide insights into the mechanisms of therapy response and resistance. Our recent genome-wide analysis of blasts isolated from peripheral blood (PB) of pediatric patients with B-cell precursor (BCP-) ALL at early stages of induction therapy (day 8) revealed an up-regulation of the gene encoding for integrin alpha M chain (ITGAM, CD11b), and pointed to the involvement of adhesion receptors in the therapy response. In the present study we investigate expression of CD11b at protein level and its clinical relevance in BCP-ALL. At diagnosis, ALL cells heterogeneously express CD11b both in PB (mean 1839+/−515 MESF, 160 pts) and in bone marrow (BM) (655+/−114 MESF, 79 pts). At day 8, increased expression of CD11b has been observed in the majority of PB samples (77 of 100 pts, up-regulation range 1.5- to 74-fold, mean 8.2-fold). In BM, the CD11b expression has been consistently up-regulated at the clinically significant MRD timepoints of induction therapy (days 15, 33 and 78; ALL-BFM 2000 protocol). Moreover, since normal CD19+/CD10+ precursor cells are CD11b negative, the detection of CD11b improved discrimination of normal and leukemic blasts in the MRD positive cases with regenerating BM at day 78. Of particular interest, evaluation of the CD11b expression at diagnosis in context of the early therapy response disclosed its potential clinical relevance. In the bivariate data analysis, expression of CD11b significantly correlated with the blast reduction rate (BRR) and blast count (BC) in PB at day 8 (p=0.007 and 0.001, 103 pts). Similarly, the CD11b expression in BM significantly correlated with the BRR and BC in BM at day 15 (p=0.024 and 0.012, 58 pts). In the multivariate regression analysis, excluding BCR-ABL and MLL-AF4 positive ALL cases and using the additional clinical parameters at diagnosis (age, white blood cell count in PB, DNA index), CD11b expression at diagnosis was the most significant variable contributing to BRR at day 8 (p<0.001, standardized beta coefficient 0.61; 98 pts) and to BRR at day 15 (p<0.001, standardized beta coefficient 0.68; 47 pts). Taken together, our data indicate that the expression of CD11b contributes to the early therapy response and has a promising potential as an MRDspecific marker in BCP-ALL.

Pharmacokinetic, pharmacodynamic and intracellular effects of PEG-asparaginase in newly diagnosed childhood acute lymphoblastic leukemia: results from a single agent window study

L-asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is thought to result from depletion of asparagine in serum and cells. We investigated the clinical response in vivo of 1000 IU/m(2) pegylated (PEG)-asparaginase and its pharmacokinetic, pharmacodynamic and intracellular effects in children with newly diagnosed ALL before start of combination chemotherapy. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common/pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (P=0.01) and BCRABL-positive ALL (P=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-asparaginase (P=0.02) and both were prognostic factors for long-term event-free survival (hazard ratio 6.4, P=0.004; hazard ratio 3.7, P=0.01). After administration of one in vivo dose of PEG-asparaginase no changes in apoptotic parameters or in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. One additional dose of PEG-asparaginase upfront ALL treatment did not lead to other severe toxicities.

Phase II Study of Combination of the HyperCVAD Regimen with Dasatinib in Patients with Philadelphia Chromosome (Ph) or BCR-ABL Positive Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Phase Chronic Myeloid Leukemia (CML-LB).

Combination of cytotoxic chemotherapy with imatinib has improved the outcome for patients with Ph+ ALL and resulted in eradication of minimal residual disease and durable remissions without allogeneic stem cell transplant in some patients (Thomas D, Blood, 2004; Yanada M, JCO, 2006; Wassmann B, Blood, 2006; de Labarthe A, Blood, 2007). The dual Src and Abl inhibitor dasatinib has a significantly higher in vitro kinase inhibition against BCR-ABL and has demonstrated potent clinical activity in patients with imatinib-resistant lymphoid blast phase CML (CML-LB) and Ph+ ALL with over 50% complete cytogenetic responses (CG CR) in phase I and II trials but with median progression free survival of only 3 to 4 months. We are conducting a phase II trial in which patients with newly diagnosed or relapsed Ph+ ALL or CML-LB receive dasatinib 50 mg po bid for the first 14 days of each of 8 cycles of alternating hyperCVAD and high dose cytarabine and methotrexate. Patients in complete remission (CR) continue to receive maintenance dasatinib 50 mg po bid daily and vincristine and prednisone monthly for 2 years followed by dasatinib indefinitely. To date 15 newly diagnosed patients with Ph+ ALL (cohort I) and 4 patients with relapsed Ph+ ALL or CML-LB (cohort II) have received a median of 4 cycles (range 1 – 8); 4 patients are receiving maintenance in CR. Median age for cohort I is 55 years (range 23 – 79) and for cohort II, 43 years (range 26 – 69); 13 and 3 patients were older than 50 years, respectively. Median WBC at diagnosis for cohort I was 4.3 × 109/L (range, 0.8 – 203.4 x 109/L). Three patients had CNS involvement. Fourteen and 3 patients in the 2 cohorts are evaluable for response to induction; 2 are too early. Thirteen patients (93%) in cohort I and all evaluable patients in cohort II have achieved CR after the first cycle; 1 patient in cohort I died on day 20 from infections before response assessment; her bone marrow exam on day 14 showed no detectable disease. Ten of 11 (91%) patients in cohort I have achieved CG CR after 1 cycle; 3 are too early. Three of 4 patients in cohort II have achieved CG CR after 1 cycle; 1 had a new CG abnormality and 1 is too early. Six patients have achieved complete molecular remission after the first cycle with the lowest BCR-ABL/ABL in the other patients ranging from 0.01 to 1.91. Median time to neutrophil and platelet recovery for cohort I is 18 and 25 days and for cohort II 18.5 and 30.5 days. Grade 3 and 4 toxicity has included 7 episodes of GI bleeding as well as infections, diarrhea, hypophosphatemia, hypocalcemia, elevated transaminases, and acute renal failure unrelated to treatment. With a median follow up of 4 months (range, 0 – 10), 15 patients are alive and in CR; 1 died at induction, 1 died in CR from an unrelated cardiac event, and 2 are too early. No patient has relapsed and no patient has received an allogeneic transplant. We conclude that the combination of the hyperCVAD regimen with dasatinib is feasible and can achieve early molecular remissions in patients with Ph+ ALL and CML-LB.

Efficacy of dasatinib in chronic phase chronic myelogenous leukemia patients after imatinib failure according to baseline BCR- ABL mutations

7023 Background: Dasatinib is an oral, multi-targeted BCR-ABL and SRC kinase inhibitor with preclinical activity against 20/21 imatinib (Im) resistant BCR-ABL mutations. Clinical efficacy was demonstrated in phase I, II, and III studies in patients (pts) with chronic myelogenous leukemia (CML) in all phases of the disease and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of BCR-ABL mutations associated with Im resistance and efficacy of dasatinib in chronic phase (CP) CML pts. Methods: Between 10/03 and 03/06, dasatinib was commenced in 1,093 CP-CML pts recruited for three consecutive trials and administered for a median of 8.7 months (range &lt;1–25.9). BCR-ABL mRNA was screened for mutations of amino acids 207–517 by D-HPLC and/or regular sequencing and data are available from 961 cases (88%). ABL polymorphisms K247R and E499E were excluded from analysis. Results: Prior to dasatinib, 75 different BCR-ABL mutations involving 56 amino acids were detected in 18/240 Im intolerant (7.5%) and 324/721 (45%) Im resistant pts. 267 pts showed one, 53 pts two, 16 pts three, and six pts four mutations. In Im resistant pts, response was not different between 370 pts with and 351 pts without baseline mutations: Complete hematologic response (CHR) was achieved in 89% vs 92%; major cytogenetic response (MCR) in 48% vs 52% being complete (CCR) in 38 vs 36%, respectively. Response dynamics were associated with preclinical activity of dasatinib: classifying mutations for IC50 values &lt;2, 2–20 and &gt;1,000nM (T315I), CHR was achieved in 93, 85 and 28%; MCR in 48, 42 and 0%; and CCR in 37, 35 and 0% of cases, respectively. During follow up, new mutations were detected in 30 cases, predominantly T315I (n=10), Y253H/F (n=4), and F317L (n=3). Conclusions: We conclude that dasatinib is capable of inducing hematologic and cytogenetic remissions in a significant proportion of Im resistant pts associated with BCR-ABL mutations, except T315I, but also in pts with BCR-ABL independent causes of resistance. Quality of response depends on the individual type of the mutation which is consistent with preclinical observations. No significant financial relationships to disclose.

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.

Diagnostic Application of Flow Cytometry in Phosphotyrosine Level Detection and Disease Status Evaluation in Patients with Bcr-abl Positive Leukemia.

Currently, karyotype analysis for chromosome translocation of 9 and 22 and FISH or gene amplification-based technique for bcr-abl fusion gene have been the major strategies for the diagnosis and disease status evaluation of bcr-abl positive leukemias. However, the method for aberranttyrosine kinase level, which is the reflection of cellular function, is limited to the detection of the level of phosphorylated tyrosine by Western blot. This method is technically laborious, costly and sometimes leads to feigned results. We thus tried to set up a flow cytometry based technique to evaluate its possible utilization in the diagnosis and the evaluation of the disease status in bcr-abl positive leukemias. Mononuclear cells from heparinized bone marrow of patients were collected. After treatment with hemolysin, 106 cells were fixated, permeabilized and then incubated with fluorescein isothiocyanate (FITC) conjugated anti-phosphotyrosine monoclonal antibody (PY20). Fluorescence conjugated microspheres were put in the system as a calibration factor in order to provide a quantitive value for the sake of comparison with different detections. The geomean value of fluorescent microsphere was adjusted to 20 in each case and the ratio of the geomean value of each detected sample to 20 was read as relative value of phosphotyrosine level. This relative value was used in statistical analysis. The human leukemia cell line Mo7e and its derivation Mo7e P210 expressing human bcr-abl fusion gene were employed as negative and positive samples to optimize the experimental conditions. Phosphotyrosine protein in fresh bone marrow cells from patients with chronic myelogenous leukemia (CML) in different phases, bcr-abl positive acute lymphoblastic leukemia (ALL) and normal controls were evaluated by semi-quantitative analysis. A total of 32 CML, including 22 in chronic phase(CP), 3 in accelerated phase(AP) and 7 in blast crisis(BC) were enrolled in this study. Four patients with bcr-abl positive ALL were also included. It was found that there was a significantly different geomean value between Mo7e and Mo7e P210. The relative values of phosphotyrosine levels were different between bcr-abl positive samples and normal controls. The value was 756±144, 865±96, 847±105 and 819±225 with CML patients in CP,AP,BC groups and ALL group respectively, while it was only 18±7 in control group. Statistical analysis showed a significant difference among these five groups. The comparison between each two groups showed a significant difference between each bcr-abl positive group and control group (P< 0.01). There were also significant differences between the AP/CML, BC/CML or ALL group and CP/CML group(P< 0.01). We observed a trend that the value of a given patient escalated with disease progression. In a patient who was on imatinib treatment for one month, there existed two peaks of cells with different fluorescent intensity, each representing a bcr-abl positive or negative cell group, coincident with the FISH result of 158 bcr-abl positive cells in 1000 detected cells. We concluded that flow cytometry analysis of the phosphotyrosine level could be used as an adjuvant technique for the diagnosis and differential diagnosis of bcr-abl positive leukemias. It may also play an important role in the evaluation of disease status or in the judgment of therapeutic efficacy during patient follow-up.

Perforin polymorphism A91V and susceptibility to B-precursor childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

Perforin plays a key role in the cytotoxicity of natural killer and cytotoxic T cells. Genetic mutations in the perforin gene (PRF1) give rise to approximately 30% cases of familial hemophagocytic lymphohistiocytosis. A frequent polymorphism, A91V (C to T transition at position 272), may impair processing of perforin protein to the active form, and has been suggested to increase susceptibility to childhood acute lymphoblastic leukemia (ALL). To investigate the role of A91V in ALL, we genotyped 2272 children with de novo ALL registered on the Pediatric Oncology Group ALL Classification study P9900 and 655 normal controls. Allele frequencies in the controls showed a very low frequency of the variant allele in blacks, 0.7% compared to 4% in white controls. In light of this, analysis was restricted to a comparison of white cases and controls only. Overall genotype frequencies were similar in white ALL cases and normal white controls (P=0.58), indicating that in contrast to the previous report, A91V polymorphism is not associated with increased risk of childhood ALL. PRF1 A91V frequency was significantly increased in children with BCR-ABL positive ALL (24 vs 8.5%; P=0.0048); however, this observation includes a relatively small number of cases and needs further exploration.

High Complete Remission Rate and Promising Outcome by Combination of Imatinib and Chemotherapy for Newly Diagnosed BCR-ABL–Positive Acute Lymphoblastic Leukemia: A Phase II Study by the Japan Adult Leukemia Study Group

A novel therapeutic approach is urgently needed for BCR-ABL-positive acute lymphoblastic leukemia (ALL). In this study, we assessed the efficacy and feasibility of chemotherapy combined with imatinib. A phase II study of imatinib-combined chemotherapy was conducted for newly diagnosed BCR-ABL-positive ALL in adults. Eighty patients were entered into the trial between September 2002 and January 2005. Remission induction therapy resulted in complete remission (CR) in 77 patients (96.2%), resistant disease in one patient, and early death in two patients, as well as polymerase chain reaction negativity of bone marrow in 71.3%. The profile and incidence of severe toxicity were not different from those associated with our historic chemotherapy-alone regimen. Relapse occurred in 20 patients after median CR duration of 5.2 months. Allogeneic hematopoietic stem-cell transplantation (HSCT) was performed for 49 patients, 39 of whom underwent transplantation during their first CR. The 1-year event-free and overall survival (OS) rates were estimated to be 60.0%, and 76.1%, respectively, which were significantly better than those for our historic controls treated with chemotherapy alone (P < .0001 for both). Among the current trial patients, the probability for OS at 1 year was 73.3% for those who underwent allogeneic HSCT, and 84.8% for those who did not. Our results demonstrated that imatinib-combined regimen is effective and feasible for newly diagnosed BCR-ABL-positive ALL. Despite a relatively short period of observation, a major potential of this treatment is recognized. Longer follow-up is required to determine its overall effect on survival.

Perforin Polymorphism A91V and Susceptibility to Childhood Acute Lymphoblastic Leukemia (ALL).

Perforin plays a key role in the cytotoxicity of NK cells and cytotoxic T lymphocytes. Genetic mutations in perforin gene (PRF1) give rise to 30–40% of cases of familial hemophagocytic lymphohistiocytosis. We recently studied two boys who had been treated for PreB ALL and subsequently developed HLH. Both showed decreased perforin expression in NK cells and were found to be heterozygous for PRF1 A91V. The functional consequences of the PRF1 A91V (resulting from C272T transition) are controversial; between 3 and 17% of apparently normal people are heterozygous for this polymorphism depending on the population studied. Recent data suggest that A91V results in conformational change that may impair processing of perforin protein to the active form, and that the polymorphism may increase susceptibility to ALL in children (Trambas et al, Blood 2005; 106:932; Santoro et al, Haematologica 2005; 90:697). We have genotyped 655 normal blood donors and 2,272 children with ALL treated on Pediatric Oncology Group 9900 clinical trials. Genotyping was performed using a specific PCR assay (Taqman). Genotypes in the normal US blood donors showed significant variation in allele frequency by race, with a very low frequency of the variant allele in black controls (0.7% compared with 4% in white controls). Allele frequencies did not vary by gender and genotype frequencies were in Hardy Weinberg equilibrium (whites C/C 92%; C/T 8%; T/T 0.2%). In light of the low frequency of the variant allele in blacks, analysis of leukemia cases was restricted to a comparison of white cases and white controls. Analyses were performed for all B-lineage ALL cases combined, and were also further stratified for known genetic subtypes of ALL.Perforin genotype CCPerforin genotype CT or TTp-value (cases versus controlsControls (n=507)464 (91.5%)43 (8.5%)-Cases (all; n= 1321)1198 (90.7%)123 (9.3%)0.58BCR-ABL22 (76%)7 (24%)0.0048Trisomy 4,10238 (93.7%)16 (6.3%)0.29TEL-AML1250 (90.6%)26 (9.4%)0.66E2A-PBX24 (92.3%)2 (7.7%)0.89MLL abn.40 (87%)6 (13%)0.29Hyperdiploid244 (92.4%)20 (7.6%)0.66Hypodiploid8 (100%)0 (0%)0.40Taken together, these data indicate that the A91V polymorphism is not associated with an overall increased risk of childhood ALL, in contrast to the previous report. PRF1 A91V was identified with significantly increased frequency in children with BCR-ABL positive ALL. However, this observation includes a relatively small number of cases and the potential association should be explored further, perhaps in adult ALL series in which the frequency of BCR-ABL positivity is likely to be high.

Combination of intensive chemotherapy and imatinib can rapidly induce high-quality complete remission for a majority of patients with newly diagnosed BCR-ABL–positive acute lymphoblastic leukemia

The outcome for adult patients with BCR-ABL-positive acute lymphoblastic leukemia (ALL) remains dismal and long-term survival can hardly be achieved except by allogeneic hematopoietic stem cell transplantation (HSCT). The Japan Adult Leukemia Study Group (JALSG) has recently started a phase 2 trial with intensive chemotherapy and imatinib for newly diagnosed BCR-AB-positive ALL patients, and we present here the interim results for the first 24 patients. All patients except one case of early death (96%) attained complete remission (CR) after a single course of remission induction therapy. Polymerase chain reaction (PCR) negativity was achieved in 28% of the patients on day 28, in 50% on day 63, and in up to 78% during the follow-up period. The toxicity profile was almost similar to that with chemotherapy alone. As a result, 15 patients (63%) could receive an allogeneic HSC transplant during their first CR. Although the number of patients is small and the observation period is too short, the combination therapy is very promising and produces high-quality CR for most newly diagnosed patients with BCR-ABL-positive ALL. This is especially useful because it provides the patients with a better chance to receive an allogeneic HSC transplant.

The Proteome Analysis of an Imatinib-Resistant Cell Line Identifies Changes in Molecular Chaperones Such as Heat Shock Proteins (Hsp): A Possible New Mechanism of Imatinib Resistance.

Targeting the tyrosine kinase activity of Bcr-Abl by imatinib mesylate is an attractive therapeutic strategy in chronic myelogenous leukemia (CML) and in Bcr-Abl positive acute lymphoblastic leukemia. However, resistance to imatinib monotherapy is currently a major issue preventing the successful treatment of CML patients. It may be mainly mediated by mutations within the kinase domain of Bcr-Abl and/or amplification of the BCR-ABL genomic locus. The K562-r imatinib-resistant cell line, derived in our laboratories from the sensitive parental K562-s has neither mechanism of resistance, nor overexpression of Src-kinases such as Lyn and Hck, as described for other cell lines. In the current study we used two-dimensional (2D) difference gel electrophoresis (DIGE) and MALDI-TOF-TOF mass spectrometry to compare the proteome of K562-r and K562-s. With the aid of the Image Master TM 2D platinium software, we detected 31 different proteins in K562-r and K562-s. These proteins were classified in 3 different groups. The first includes proteins involved in the synthesis and stability of RNA (hnRNP K, hnRNP H, CstF , transcription elongation factor A protein 1, PCBP2, TCP1), the second encompasses structural proteins (CAPG, fascin, tubulin, vimentin, laminA, C tubulin beta-1 chain, actin cytoplasmic 1, keratin type I type II), and the third was represented by different enzymes participating in general metabolic pathways (glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, mitochondrial precursor, glutamate dehydrogenase 2, pyruvate kinase, PURH protein). Furthermore, chaperone proteins such as heat-shock protein Hsp60, P60HOP or STI-1, Hsp105 and Hsp70 were differentially expressed in the sensitive and resistant cell lines. Since these proteins complex with Hsp90 and this complex has been reported to interact with the Bcr-Abl protein, we focused on these molecular chaperones. Hsp70 family proteins such as Hsc70 and Hsp74 were found to be more expressed 2.5-fold higher in K562-r than in K562-s, and/or exhibited post translational modifications (phosphorylation and acetylation) confirmed by Western blotting. Hsp70 was recently described as an inhibitor of apoptosis (Ray S et al., JBC 2004) and its overexpression in K562-r could thus contribute to its imatinib-resistant phenotype. Preliminary functional studies showed that whereas K562-s and K562-r were equally sensitive to the apoptotic effect of geldanamycin (an inhibitor of Hsp90), the combination of geldanamycin and a proteasome inhibitor (MG132) was more efficient in K562-r than in K562-s (viability of 16% and 40% respectively after 4 days in culture). Ongoing experiments utilizing siRNA against Hsp70 will help understand the link between the expression profile of Hsp proteins and the imatinib-resistant phenotype of this cell line. In conclusion, the use of a new experimental strategy, i.e. proteomic analysis by DIGE and mass spectrometry, allowed us to identify selected proteins whose patterns of expression and post-translational modification may underlie a new mechanism of resistance to imatinib in Bcr-Abl positive cells.

Combination of Intensive Chemotherapy and Imatinib (IDEAMOP Regimen) for the Treatment of Newly Diagnosed BCR-ABL Positive Acute Lymphoblastic Leukemia; Excellent Efficacy without Increasing Toxicity.

The Philadelphia chromosome, which constitutes BCR-ABL fusion gene, is the most frequent cytogenetic abnormality in adult acute lymphoblastic leukemia (ALL). Although complete remission (CR) is achieved in 50–80% of patients, most of them suffer a relapse, resulting in overall survival (OS) of approximately 10% by standard chemotherapy. Recently, the Japan Adult Leukemia Study Group (JALSG) has started a phase 2 trial for newly diagnosed BCR-ABL positive ALL patients, and 60 patients have been enrolled at this time. We present here the results for the first 41 patients. After screening for BCR-ABL at presentation, those with positive BCR-ABL were treated with IDEAMOP regimen. For remission induction therapy, intensive chemotherapy comprising daunorubicin, cyclophosphamide, vincristine (VCR) and predonisolone (PSL) was combined with imatinib at a dose of 600 mg/day administered from day 8 to day 63. Consolidation therapy consisted of the odd courses with high-dose methotrexate and high-dose cytarabine and the even courses with 600 mg/day of imatinib alone for 28 days, which were repeated alternatively for 4 cycles. Thereafter, patients received maintenance therapy with VCR, PSL and imatinib until 2 years from the date they had attained CR. If an HLA-identical sibling donor was available, allogeneic hematopoietic stem cell transplantation (HSCT) was recommended. HSCT from an alternative donor was used at the discretion of the institution. Between September 2002 and March 2004, a total of 41 patients with newly diagnosed BCR-ABL positive ALL were treated with IDEAMOP. All patients except two cases of early death (95%) attained CR. PCR negativity in bone marrow was achieved in 25% of the patients on day 28, in 57% on day 63, and in up to 73% during the follow-up period. Toxicity of the remission induction therapy was almost similar to that observed for chemotherapy alone. The median time of WBC recovery to at least 1,000/mL was 19 days (range, 14 to 29 days), and the median time of platelet recovery to at least 100,000/mL was 22 days (range, 14 to 30 days). Death occurred in two patients, one for pulmonary bleeding on day 10, and the other for pneumonia on day 32. Interruption of imatinib was implemented for 11 patients (27%) for the following reasons; nausea in 4, GPT elevation in 3, pulmonary bleeding, pneumonia, fluid retention, ileus, amylase elevation in one patient, respectively; however, none of the 39 surviving patients dropped out the protocol due to intolerance of treatment. Four patients had a relapse during consolidation therapy after 4.0, 4.5, 4.9 and 10.3 months of CR. Allogeneic HSCT was performed for 20 patients (9 from a sibling donor, 8 from an unrelated donor, and 3 from unrelated cord blood) during their first CR. The 1-year event-free survival (EFS) and OS rates were estimated at 78% and 88%. Outcomes were superior to those in ALL93 study, where patients were treated with chemotherapy alone, in terms of CR rate, EFS, and OS (P < 0.0001, P < 0.0001, and P = 0.0118, respectively). In summary, our study demonstrated that IDEAMOP produces high-quality CR for a majority of patients with newly diagnosed BCR-ABL positive ALL without an increase in toxicity. This is especially useful for providing patients with a better chance to receive allogeneic HSCT. Long-term efficacy will be addressed in the final analysis of this study.

Generation of Resistance Cell Lines to AMN107, a New Inhibitor of BCR-ABL and Its Effects on Cell Lines Sensitive and Resistant to Imatinib.

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myelogenous Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Imatinib is a selective inhibitor of Bcr-Abl tyrosine kinase and is now used in frontline therapy for CML. However clinical resistance is the main concern using this treatment, mediated by mutations within the kinase domain of Bcr-Abl, amplification of the BCR-ABL genomic locus or other as yet unknown mechanisms. AMN107 (Novartis Pharma AG, Basel, Switzerland) is a synthetic, second generation inhibitor of Bcr-Abl tyrosine kinase. In the current study, we tested AMN107 against different Bcr-Abl positive cell lines such as K562, LAMA84, AR230 or murine Ba/F3 cells transfected with BCR-ABL (BaF/BCR-ABL). In a 4 day proliferation assay (MTS) the dose of AMN107 that inhibited 90% of the cells (IC90) was 0.01 μM which was 100-fold lower than the IC90 of imatinib. In addition, proliferation of imatinib resistant cell lines which exhibited amplification of BCR-ABL was inhibited by 75% in the presence of 0.01 μM AMN107. However, Ba/F3 cells expressing the imatinib resistant BCR-ABL T315I mutant were only inhibited with 10μM of AMN107. Furthermore, K562-R, an imatinib-resistant cell line exhibiting a new mechanism of imatinib resistance (modification of chaperone proteins such as heat shock proteins) was also insensitive to AMN107; the IC90 for AMN107 at day 4 was 1μM versus 4μM for imatinib.Finally, we investigated potential resistance to AMN107 in Bcr-Abl positive cells. Resistant cell lines were generated after long-term (2 month) gradual dose-escalation exposure to the inhibitor. Up to now, we have obtained four cell lines, AR230-ra, K562-ra, LAMA 84-ra, and BaF/BCR-ABL-ra resistant to 8, 10, 10, and 100 nM of AMN107, respectively. Resistance was defined as the capacity to survive in the continuous presence of doses of AMN107 that kill in three days more than 90% of the parental cells in liquid culture.Preliminary investigations of AMN107 resistance using western blot and cytometry have shown that only BaF/BCR-ABL-ra overexpressed wildtype Bcr-Abl as we have already reported for imatinib resistance. Studies with these resistant cell lines investigating cross resistance with imatinib and looking for additional mechanisms of resistance are in progress.We conclude that in vitro, AMN107 is more powerful than imatinib in inhibiting the proliferation of BCR-ABL positive cell lines. In addition, we have demonstrated that it is possible to develop resistant cell lines to this new inhibitor of Bcr-Abl.

633. Dose-Dependent Gene Silencing by Lentivirus-Mediated RNA Interference (RNAi) in bcr-abl Positive Cells

RNA interference (RNAi) describes a process of double-stranded RNA-dependent post-transcriptional gene silencing. This gene silencing is mediated by 21–23 nt small interfering RNAs (siRNAs) which induce sequence-specific mRNA degradation. Fusion-transcripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi-approach. Recently, siRNAs have been shown to specifically inhibit expression of the bcr-abl oncogene essential for chronic myeloid and bcr-abl positive acute lymphoblastic leukemia (CML, ALL) in hematopoietic cell lines. Since RNAi triggered by a single siRNA application is only transient in mammalian cells potential anti-tumor effects are limited in the models studied so far. Gene transfer of transcription cassettes for suitable RNAi-triggers, however, can induce long-term gene silencing even in primary hematopoietic cells. Using lentiviral gene transfer to transcribe different shRNAs targeting the bcr-abl oncogene and to express red fluorescence protein (RFP) as a quantitative reporter to track cellular transduction we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+human K562 and murine TonB cells. Importantly, killing of bcr-abl+cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced cells. Furthermore, we show that lentiviral transduction of homogenous cell lines results in heterogenous cell populations in terms of lentiviral integrations, RFP-and siRNA expression, target gene silencing, and functional phenotype. Whereas transduced K562 cells with high RFP-expression are eradicated, a limited number of cells with lower RFP-expression and smaller number of lentiviral integrations may outgrow after longer periods of cell culture. Interestingly, these cells still show some reduction in bcr-abl mRNA levels, aberrant proliferation kinetics, and higher sensitivity to the Bcr-Abl kinase inhibitor STI571 as compared to controls. Furthermore, lentiviral gene transfer of anti bcr-abl shRNA into primary CML cells can inhibit colony formation of transduced CD34+progenitor cells by about 50%. These data demonstrate that lentivirus-mediated RNAi can induce high efficient and stable gene silencing depending on the number of lentiviral integrations in cell lines and in primary cells. Selection processes of gene modified cells, however, have implications for any potential therapeutic application and for functional genomic analysis based on gene transfer strategies to induce stable RNAi in mammalian cells.

Expression of BCR-ABL, E2A-PBX1, and MLL-AF4 Fusion Transcripts in Newly Diagnosed Children with Acute Lymphoblastic Leukemia: A Children's Cancer Group Initiative

We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2APBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Children's Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.