ALBERT

Description: The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph+ ALL were analyzed by direct sequencing of a 714–base pair region of ABL encoding for the adenosine triphosphate (ATP)–binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph+ ALL samples resistant to STI571 have a unique mutation Glu255Lys of BCR-ABL.

Description: The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15RT) which was derived from the previously well characterized SupB15 cell line (SupB15WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15RT cells. Thus, the SupB15RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15RT cells, in contrast to SupB15WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15WT cells by modulation of growth, survival and migration. When SupB15WT cells were treated with LPA, alone or in combination with imatinib, SupB15WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15RT vs. SupB15WT cells. We found no influence on apoptosis in imatinib treated SupB15WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.

Description: Patients with CML in lymphoid blast crisis (LBC-CML) or advanced Ph+ ALL have an unsatisfactory and only brief response to imatinib mesylate (IM). Moreover, treatment options in pts who failed IM are extremely limited. Dasatinib (BMS-354825) is a novel, oral kinase inhibitor that targets BCR-ABL and SRC kinases, and has shown promising clinical activity in a Phase I dose escalating study in patients with BCR-ABL-positive leukemias. Between January 2005 and June 2005, 77 pts (42 CML-LBC and 35 Ph+ ALL) who had failed IM-based therapy were enrolled in this multinational Phase II study investigating the safety and efficacy of dasatinib. This preliminary analysis summarizes data on the first 28 pts accrued (13 CML-LBC and 15 Ph+ ALL) who were accrued prior to March 20, 2005. Dasatinib was administered orally at 70 mg twice daily (BID) on a continuous daily dosing schedule; dose escalation to 100 mg BID or dose reduction to 50 mg and 40 mg BID were allowed for poor initial response or persistent toxicity, respectively. Complete blood counts were performed weekly and bone marrow evaluation, including cytogenetic analysis, was scheduled every month. Mutation analyses were performed in all pts. 27 pts were IM resistant and 1 was IM intolerant; 17 (61%) pts had received prior IM doses 〉600 mg/day, 13 (46%) pts received IM for

Description: Background: Nilotinib is a novel orally active aminopyrimidine. It is a highly specific Bcr-Abl tyrosine kinase inhibitor (TKI) 30-fold more potent than imatinib. Based on the efficacy and favorable tolerability demonstrated by nilotinib in the phase I study, this phase II open-label study was designed to evaluate the safety and efficacy of nilotinib in adult pts with Ph+ chronic myeloid leukemia in blast crisis (CML-BC) resistant to or intolerant of imatinib. The prognosis for these pts remains poor. Methods: The primary endpoint was confirmed hematologic response (HR). Imatinib resistance was defined as either treatment with imatinib 〉600 mg/day with disease progression, no HR in bone marrow after 4 weeks, or pts receiving 1 month while on imatinib. Nilotinib therapy was commenced at 400 mg twice daily (BID) with escalation to 600 mg BID for pts who had inadequate responses and no safety concerns. Results: Safety and efficacy data are reported for 135 BC (myeloid, n=103; lymphoid, n=29; unknown, n=3). The median age was 55 (18–79) years, the median time since CML diagnosis was 1.6 (/=95% Ph+ metaphases at study entry. Chromosomal abnormalities other than Ph+ were also noted in 54% of pts at study entry. Extramedullary involvement was present in 39% of the pts. The most common Grade 3/4 hematologic laboratory abnormalities were neutropenia (67%), thrombocytopenia (62%), anemia (42%). The most common Grade 3/4 non- hematologic AEs were as follows: pneumonia (11%), pyrexia (7%), nausea (4%), diarrhea (4%), and asthenia (4%). Conclusions: Based on the CHR rates achieved in this very advanced patient population, nilotinib monotherapy appears to have clinical activity in pts with imatinib-resistant CML BC. Overall nilotinib is well tolerated in this patient population with advanced disease and with non-heme toxicity comparable to that observed for pts with CML-CP. The hematological responses were similar between myeloid and lymphoid Ph+ CML blast crises. Response for Patients With CML-BC With at Least 6 Months of Follow-up Myeloid (N = 103) n (%) Lymphoid (N = 29) n (%) Hematologic response 40 (39) 11 (38) CHR 25 (24) 8 (28) Marrow response 5 (5) 1 (3) Return to chronic phase 10 (10) 2 (7) SD 31 (30) 7 (24) PD 18 (17) 6 (21) Not evaluable 5 (5) 1 (3) Missing 9 (9) 4 (14)

Description: Introduction: Febrile neutropenia is a common and potentially fatal consequence of Acute Myeloid Leukemia (AML). We performed a retrospective analysis of 97 adult AML patients (median age 67.6 y (58–78) treated between February 2000 and June 2005. Aim of the study was to evaluate risk factors for infectious complications and to identify underlying causative pathogens during first and second cycle of intensive induction chemotherapy. For statistical evaluation of the results, Students t-test, Fisher’ exact test or Chi square test were performed as appropriate. Results: Of the 97 episodes 64% occurred in male patients at a median age of 67.6 years (58–78). Patients developed fever not related to cytotoxic therapy or transfusion in 77 (80%) and no fever in 20 (20%) of the episodes. Compared to patients without infectious complications patients with fever had significantly more pretreatment comorbidities (0.80±1.20 vs 1.81±1.41, p=0.0042), a prolonged period of severe neutropenia (

Description: Acute promyelocytic leukemia (APL) is distinguished from other AMLs by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17) which leads to the expression of the PML/RAR fusion protein. PML/RAR is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA). Patients suffering from APL undergo complete hematologic but not molecular remission upon treatment with t-RA. Virtually all patients treated with t-RA-monotherapy had a rapid relapse within few months. But in the combination with an anthracycline, such as doxorubicin or idarubicin, t-RA improved the long term outcome of APL-patients dramatically. Nothing is known about why t-RA-monotherapy is unable to eradicate completely the leukemic population and how it increases the response to chemotherapy. In vitro, the exposure of early hemopoietic stem cells (HSCs) to t-RA does not induce differentiation but selects immature progenitors. Moreover, mice lacking the t-RA-specific receptor RARalpha do not exhibit an impairment of granulopoiesis or hemopoiesis. The indication, that t-RA may be involved in the hemopoietic differentiation, is given by the HL-60 cell line which undergoes granulocytic differentiation at the pharmacological dosages (10−6M) of t-RA. Furthermore vitamin A-deficient mice or mice treated with a antagonist of t-RA accumulate more immature granulocytes in the bone marrow. PML/RAR mediates the response of APL blasts to t-RA, but it is completely unclear, which effect t-RA exerts on the PML/RAR-positive leukemic stem cells which maintains the blast population and represents the source of relapse. Therefore we investigated the effect of t-RA on a cell population with stem cell capacity expressing PML/RAR isolated from the APL cell line NB4 as well as from CD34+/CD38- KG-1 cells transfected with PML/RAR. Here we report that i) the NB4 cells engrafted in NOD/SCID mice indicating the presence of a subpopulation with stem cell capacity in NB4 cells; ii) NB4 had a Hoechst 3342 excluding side population (SP) representing about 1% of the whole cell population; iii) t-RA reduced but did not deplete the side population in NB4 cells; iv) the expression of PML/RAR increased CD34+/CD38- population in KG-1 cells from 75% to over 95%; v) t-RA reduced the CD34+/CD38- population from 75% to 3,5% in mock transfected KG-1 confirming its capacity to induce differentiation, whereas in PML/RAR-positive KG-1 cells it led only to a reduction from 98% to a 25%, which still maintain the capacity to engraft in NOD-SCID mice; vi) also the expression of other fusion proteins, such as AML-1/ETO or PLZF/RAR, associated with t-RA-resistant AML-subtypes, increased the percentage of CD34+/CD38- KG-1 cells over 90%, which was reduced by t-RA only to 35% and 19%, respectively. Taken together these data suggest that a subset of early HSC expressing PML/RAR exhibit the same t-RA-resistant phenotype as HSC expressing fusion proteins associated with AML-subtypes which, in contrast to APL, do not respond to t-RA. These data may give an explanation, why APL-patients do not achieve complete molecular remission upon t-RA monotherapy and undergo early relapse.

Description: In adult Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL), minimal residual disease (MRD) after stem cell transplantation (SCT) is associated with a relapse probability exceeding 90%. Starting imatinib in the setting of MRD may decrease this high relapse rate. In this prospective multicenter study, 27 Ph+ ALL patients received imatinib upon detection of MRD after SCT. Bcr-abl transcripts became undetectable in 14 (52%) of 27 patients, after a median of 1.5 months (0.9-3.7 months) (earlyCRmol). All patients who achieved an earlyCRmol remained in remission for the duration of imatinib treatment; 3 patients relapsed after imatinib was discontinued. Failure to achieve polymerase chain reaction (PCR) negativity shortly after starting imatinib predicted relapse, which occurred in 12 (92%) of 13 patients after a median of 3 months. Disease-free survival (DFS) in earlyCRmol patients is 91% ± 9% and 54% ± 21% after 12 and 24 months, respectively, compared with 8% ± 7% after 12 months in patients remaining MRD+ (P 〈 .001). In conclusion, approximately half of patients with Ph+ ALL receiving imatinib for MRD positivity after SCT experience prolonged DFS, which can be anticipated by the rapid achievement of a molecular complete remission (CR). Continued detection of bcr-abl transcripts after 2 to 3 months on imatinib identifies patients who will ultimately experience relapse and in whom additional or alternative antileukemic treatment should be initiated.

Description: Background: ABL001 is a potent, specific BCR-ABL inhibitor in development for the treatment of patients with CML and Ph+ ALL. ABL001 binds a pocket on the BCR-ABL kinase domain normally occupied by the autoregulatory myristoylated N-terminus of ABL1, which is lost upon fusion with BCR. ABL001 was designed to inhibit BCR-ABL in a non-ATP-competitive manner to maintain activity against BCR-ABL mutations that confer resistance to TKIs. Preclinically, combined with ATP-competitive TKIs, ABL001 eliminates early leukemic progenitors and diminishes emergence of resistant clones. Methods: Patients with CML-CP, -AP, or -BP with failure of ≥ 2 prior TKIs and Ph+ ALL patients with failure of ≥ 1 prior TKI due to resistance or intolerance were enrolled in this first-in-human, multicenter, open-label phase 1 dose-escalation study (CABL001X2101). Escalating doses of single-agent ABL001 were administered orally on continuous twice-daily (BID) or once-daily (QD) schedules or in combination with imatinib, nilotinib, or dasatinib. Therapy continued until disease progression, unacceptable toxicity, consent withdrawal, or death. Primary objectives were to estimate the maximum tolerated dose (MTD) of ABL001 administered as single agent or combined with TKIs. Secondary objectives included safety, preliminary efficacy, and pharmacokinetics of ABL001 alone and combined with TKIs. Dose escalation followed a Bayesian logistic regression model based on dose-limiting toxicities (DLTs) in cycle 1. Results: At data cutoff, 101 CML and Ph+ ALL patients had received ABL001 as either single agent or in combination. 67 CML and 6 Ph+ ALL patients have been treated at single-agent BID doses: 10 mg (n = 1), 20 mg (n = 14), 40 mg (n = 30), 80 mg (n = 14), 150 mg (n = 9), 200 mg (n = 5), with MTD not reached. A dose of 40 mg BID has been recommended for CML-CP patients. 19 CML patients have been treated with single-agent QD doses: 80 mg (n = 3), 120 mg (n = 10), 200 mg (n = 5), with MTD not yet reached. 9 CML patients have been treated in combination with TKIs with dose escalation ongoing. Median age was 55 y (range, 23-79 y). Most patients (98%) had baseline ECOG status 0-1. Patients were heavily pretreated; 65 (65%) had received 〉 2 prior TKIs. 70 patients were resistant to their last TKI. In 84 patients, treatment is ongoing at doses 20-200 mg BID or QD and at respective combination doses, with 17 patients discontinued (disease progression [n = 6], AEs [n = 7], consent withdrawal [n = 4]). Median duration of exposure is 34 wk (range, 0-98 wk). ABL001 pharmacokinetics was dose-proportional with minimal accumulation across dose and time. There were 5 DLTs: 2 Gr 3 lipase elevations (40 mg BID and 200 mg QD), 1 Gr 2 arthralgia (80 mg BID), 1 acute coronary syndrome (150 mg BID), and 1 Gr 3 bronchospasm (200 mg BID). 3 cases of Gr 2 acute pancreatitis occurred in cycle 5 at doses ≥ 80 mg BID. The majority of AEs regardless of study drug relationship were grade 1/2. Most common grade 3/4 AEs suspected related to ABL001 were lipase increase (8%), thrombocytopenia (7%), anemia (5%), and neutropenia (4%). 1 non-study drug-related death due to multiorgan failure occurred. 55 patients (10-200 mg BID) on therapy for ≥ 3 months had efficacy assessments. 7/9 patients in cytogenetic relapse (〉 35% Ph+ metaphases at baseline) achieved CCyR by 6 months, with all 7 maintaining CCyR by 12 months. 13 of 55 (23.6%), 16 of 37 (43.2%), and 20 of 55 (57.1%) patients achieved or maintained MMR by 3, 6, and 12 months, respectively. Of 47 patients with baseline BCR-ABL 〉 0.1 % IS, 6 of 47 (12.8%), 9 of 30 (30%), and 13 of 28 (46.4%) patients achieved MMR by 3, 6 and 12 months, respectively. Of 32 patients with baseline BCR-ABL ≤ 10 % IS, 4 (12.5%), 9 (28%), and 11 (34%) achieved ≥ 1-log reduction of BCR-ABL % IS by 3, 6 and 9 months, respectively, with all maintained at 12 months. 17/20 patients who achieved MMR have maintained it. Clinical activity was seen in multiple TKI-resistant mutations. 7 patients with T3151I were enrolled: 3 achieved CCyR (1 relapse by 6 months) and 1 maintained baseline MMR at median follow-up of 8 months. Only 1/6 relapsed patients had detectable myristoyl-pocket mutations (V468F, I502L). Conclusion: ABL001 appears well tolerated to date and exhibits significant and durable activity in a heavily pretreated subgroup of CML patients. 40 mg BID has been recommended for CML-CP patients. Phase 1 accrual is ongoing for CML patients on QD or combination therapy, with T3151I mutations, and Ph+ ALL patients. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Goh:Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Fusion Pharma: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Minami:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Rea:Ariad: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Lang:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mauro:Ariad: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Pfizer: Consultancy. Talpaz:Incyte Corporation: Other: Travel expense reimbursement, Research Funding; Novartis: Research Funding; Ariad: Other: Expense reimbursement, travel accomodation expenses, Research Funding; Pfizer: Consultancy, Other: travel accomodation expenses, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Ariad: Research Funding. Breccia:Pfizer: Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Novartis: Consultancy, Honoraria. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Heinrich:MolecularMD: Consultancy, Equity Ownership; Novartis: Consultancy, Honoraria, Patents & Royalties; Ariad: Consultancy. Janssen:Pfizer: Honoraria; Novartis: Research Funding; Ariad: Honoraria; BMS: Honoraria. Steegmann:Pfizer: Honoraria, Other: research funding for the Spanish CML group; Novartis: Honoraria, Other: research funding for the Spanish CML group; BMS: Honoraria, Other: Research funding for the Spanish CML Group; Ariad: Honoraria, Other: Research funding for the Spanish CML Group. Mahon:Ariad: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Duan:Novartis Institutes for Biomedical Research, Inc.: Employment. Iyer:Novartis Institutes for Biomedical Research, Inc.: Employment. Hynds:Novartis Institutes for Biomedical Research, Inc.: Employment. Vanasse:Novartis Institutes for Biomedical Research, Inc.: Employment. Kim:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; II-Yang: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.