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M. Mohammed, S. Shin, S. Deng, J. Ford, R.L. Paquette, C.L. Sawyers, N. Rao (Intr. by Charles L. Sawyers) Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA; Novartis Pharmaceuticals, Basle, Switzerland A phase I clinical trial of the Abl-specific kinase inhibitor STI571 has shown significant activity in chronic myelogenous leukemia (CML) blast crisis and Ph+ acute lymphocytic leukemia (ALL), but a substantial fraction of these patients relapsed on drug after obtaining partial or complete responses (Druker et al ASH: 697a,1999). The mechanism of STI571 resistance is unknown. Prior work has shown that some Ph+ cell lines cultured in vitro for prolonged periods in low doses of STI571 can develop resistance to standard doses. In one instance this was associated with amplification of the BCR/ABL fusion gene (le Coutre et al, Blood 95:1758, 2000). Here we report a similar event in three patients enrolled on phase I or II multicenter trials of STI571, one with ALL blast crisis and two with advanced CML, all of whom developed relapsed leukemia after an initial response. After 3 months of STI571 treatment, an unusual marker chromosome developed in addition to the typical Ph in two of the patients. The marker appears to be a der(22)inv dup(22)(q11.2) t(9;22)(q34;q11.2), essentially a duplicated, inverted Ph. As STI571 treatment progressed, cytogenetic and FISH analyses demonstrated that in both patients, this marker chromosome appeared to be unstable, presenting in three-eight copies and in some cells forming ring chromosomes. FISH with a dual-color BCR/ABL probe illustrated that these ring chromosomes contained multiple copies of the BCR/ABL fusion gene. As STI571 treatment progressed in the third patient, rather than increases in the copy number of the marker Ph, there appeared to be a tandem amplification of the duplicated inverted Ph leading to a marker chromosome with several copies of the BCR/ABL fusion gene. The percentage of cells containing the Ph marker chromosome increased as STI571 treatment progressed, indicating selection for this aberrant clone in the three patients. Of note, there was no evidence of the duplicated inverted Ph marker chromosome or BCR/ABL fusion gene amplification in 21 other CML blast crisis or Ph+ ALL patients who relapsed on STI571 treatment. These data are the first to establish a cytogenetically observable BCR/ABL gene amplification in patients treated with STI571 and provide an explanation for drug resistance in these cases. Early detection of this marker may identify patients predisposed to BCR/ABL gene amplification, and hence resistance, when treated with STI571.
Albert B. Deisseroth, Tao Wang, Enrica Lerma, Fei Liu, Xiang-Yang D. Guo, Takuma Fujii, David Austin Yale University School of Medicine, New Haven, CT The presence of the p210bcrabl kinase leads to the phenotype of growth factor independent and anchorage independent growth in chronic myelogenous leukemia (CML). Druker and his collegues have reported that kinase inhibitory compounds which bind to the ATP binding site of the abl kinase lead to reduction of the level of circulating leukemia cells in CML and cytogenetic remissions in selected patients. In our laboratory, computational analysis of the gamma phosphate and adenosine moieties of ATP led to the design of a bicyclic scaffold and a furan planar scaffold on which can be displayed chemical functionalities which bind to local regions of the p210bcrabl kinase pocket. We tested these compounds in cell free assays of abl kinase inhibition, and assays of proliferation inhibition in suspension of the 32DP210bcrabl cell line, derived from the myeloid leukemia cell line 32D. 32D is dependent on IL3 for proliferation and anchorage independent growth, while the cell line p210bcrabl is independent of IL3 for growth in suspension and methyl cellulose. Structure activity studies of the bicyclic scaffold have resulted in mimics of both the adenosine and the triphosphate which direct the scaffold to compete with ATP for binding in the ATP binding site of the p210bcrabl kinase. Kinase inhibition studies of 27 furan planar scaffold molecules, each with a different manually synthesized substitutent ring and branched chain substitutent chemical functionalities on the furan ring structure have shown inhibition of the abl kinase in a stereoisomeric specific manner in cell free kinase inhibition assays. In addition, a cell based assay involving plating of the 32DP210bcrabl cell line at 5 cells per microwell in serum free medium, exposure of the cells to the furan inhibitory compounds for 15 minutes, addition of serum and incubation for 10 days have shown a profile of inhibition in the micromolar range which is identical to the results in the cell free kinase inhibition assay. These compounds are undergoing study in the 32DP210bcrabl cell line in the methylcellulose culture prior to proceeding to a combinatorial diversification of the scaffold molecules.These results suggest that the furan scaffold will be a useful building block for the generation of a new family of p210bcrabl kinase inhibitors.
Mercedes E. Gorre, Kimberly Banks, Nicholas C. Hsu, Monsour Mohammed, Nagesh P. Rao, John M. Ford, Ronald L. Paquette, Charles L. Sawyers Department of Medicine, University of California Los Angeles; Department of Pathology and Laboratory Medicine, University of California Los Angeles; Molecular Biology Institute, University of California Los Angeles; Novartis Pharmaceuticals, Basel, Switzerland Phase I clinical trials of the Abl-specific kinase inhibitor STI571 have shown remarkable activity in chronic phase CML, blast crisis and Ph+ acute lymphocytic leukemia (ALL) (Druker et al ASH: 368a, 697a, 1999). Whereas remissions in chronic phase patients are durable, many patients with advanced disease have relapsed on STI571 after obtaining partial or complete responses. Advanced stage CML is characterized by multiple cytogenetic and molecular abnormalities in addition to the Bcr-Abl translocation. Therefore, a central issue regarding the mechanism of resistance is to distinguish between leukemogenesis mediated by Bcr-Abl versus other oncogenic pathways. We addressed this question in 18 patients with advanced Ph+ leukemias treated on phase I or II multicenter trials of STI571, who relapsed on drug after an initial response. Bcr-Abl kinase activity in blood or bone marrow cells was measured during the course of treatment using a quantitative immunoblot assay for Crkl, a direct substrate of Bcr-Abl. Phosphorylated Crkl is detected in cell lysates as a band shift in SDS-PAGE -- providing a highly reproducible, one step method for monitoring Bcr-Abl kinase activity in patient material. Crkl phosphorylation decreased 0.5 to 7-fold in patients responding to STI571. However, phosphorylated Crkl reappeared at the time of relapse in 17 of 18 patients (5/5 Ph+ ALL and 12/13 CML blast crisis), indicating reactivation of Bcr-Abl kinase in these patients cells. This result was confirmed by phosphotyrosine immunoblot. Relapsed cells remained sensitive to STI571 ex vivo, although up to 5-fold higher doses (1.0-10.0 uM) were required when compared to cells from the same patient obtained prior to treatment initiation. In 2/18 cases, this increased STI571 dose requirement was associated with approximately 20-fold amplification of the Bcr-Abl fusion gene and a cytogenetically aberrant, duplicated inverted Ph chromosome. These studies establish that STI571 resistance in relapsed Ph+ leukemia is associated with restoration of Bcr-Abl signaling, indicating that Bcr-Abl remains a valid therapeutic target in these patients. Higher dose STI571 treatment may be indicated in this setting.
Neil P. Shah, David S. Snyder, John M. Nicoll, Ross J. McMahon, Nicholas C. Hsu, Stephen J. Forman, John M. Ford, Charles L. Sawyers, Ronald L. Paquette Medicine, Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, CA; Medicine, Division of Hematology, City of Hope National Medical Center, Duarte, CA; Novartis Pharmaceuticals, Basel, Switzerland STI571 is a novel tyrosine kinase inhibitor with relatively specific activity against the Bcr-Abl fusion protein. As previously reported [Druker et al, Amer Soc Hem 3082 (1999) abstract] STI571 has activity in Philadelphia chromosome-positive leukemias. Our institution is participating in large international multicenter studies of STI571 in chronic and advanced stage chronic myelogenous leukemia (CML). In clinical trials, we have observed 20 cases of cytogenetic remission in response to STI571 as monotherapy, as assessed by both karyotype and fluorescence in situ hybridization (FISH). In an effort to determine if minimal residual disease is detectable by the most sensitive molecular methods currently available, we have extracted RNA from leukocytes of patients who have no evidence of the Philadelphia chromosome by FISH. Using a reverse transcriptase polymerase chain reaction (RT-PCR) assay, we have identified six individuals in whom the Bcr-Abl transcript cannot be detected at a sensitivity of 1/105 cells including two patients who were in accelerated phase and one who was in blast crisis at the time therapy was instituted. As an internal control, c-Abl expressed from unrearranged chromosome 9 was detected by RT-PCR in all samples, attesting to the quality of the RNA utilized in this analysis. Furthermore, the fusion Bcr-Abl transcript was detected in all patients tested who had cytogenetic evidence of disease. The ability to assess for molecular remission by RT-PCR may be of great assistance in guiding clinical decisions in cases of complete cytogenetic remission. For patients who have evidence of disease only by RT-PCR, a quantitative assay is currently being used to assess tumor burden. Quantitative RT-PCR will provide the only means of staging disease in this subgroup of patients.
B. Wassmann, U. Scheuring, Ch. Thiede, M. Bornhäuser, F. Griesinger, E. Petershofen, H. Gschaidmeier, R. Capdeville, D. Hoelzer, O.G. Ottmann A 25-yr.-old male with Ph-pos. CML and early onset lymphoid blast crisis relapsing after a 2nd non-myeloablative allogeneic, HLA-identical sibling PBSCT despite grade III GvHD (gut, skin) was referred to our hospital for treatment with the ABL-tyrosine kinase inhibitor STI571 within a multicenter phase II clinical trial (STI109) in October 1999. Previous phase I clinical trials of STI571 have shown remarkable activity in chronic phase CML, blast crisis and Ph+ acute lymphocytic leukemia (ALL) (Druker et al ASH: 368a, 697a,1999). The patients medical history included a 7-month iv. drug abuse, acute hepatitis B infection 2 yrs. prior to diagnosis of CML and ongoing methadone substitution. Baseline cytogenetics revealed complex aberrant karyotype including t(9;22) in 83% of metaphases, bone marrow analysis showed marked hypercellularity and accelerated phase of CML, donor chimerism had dropped to 76%. STI571 therapy was initiated at a single daily dose of 400 mg p.o.. GvHD prophylaxis with steroids 60mg/d was tapered and discontinued after 3 months without recurrence of GvHD. After 4 wks. treatment marrow cytology normalized, a complete cytogenetic response and an increase in donor chimerism to 94% at 4 wks. and to >99% at 9 wks. occurred. BCR-ABL expression as measured by real time quantitative PCR showed a decrease by more than 2 logs after 4 wks. of STI571 treatment and remained negative since 8 wks. after starting treatment. The negative values reflect an overall reduction of BCR-ABL expression by more than 4 logs. Complete cytogenetic and molecular remission and stable donor chimerism are maintained after 9 mts. of treatment. STI571 was well tolerated, treatment related side effects were limited to reversible grade II neutropenia and grade I nausea not requiring pharmacologic intervention. Reactivation of hepatitis B after 7 mts. of treatment with rapid increase in liver enzymes necessitated short-term interruption of therapy and initiation of antiviral therapy. The pronounced clinical efficacy of STI571 as seen in this pt. demonstrates that STI571 is a promising therapeutic option in patients with BCR-ABL positive leukemias who have failed allogeneic bone marrow transplantation. Our findings provide the rationale for a novel treatment strategy employing STI 571 subsequent to allogeneic bone marrow transplantation.
K. Karamlou, L. Lucas, B. Druker Leukemia Program, Oregon Health Sciences University, Portland, OR, USA The Bcr-Abl oncogene is a constitutively activated tyrosine kinase and the causative agent of chronic myelogenous leukemia (CML). STI571, a tyrosine kinase inhibitor specific for the Abl tyrosine kinase, is currently in clinical trials for the treatment of CML and shows significant activity with minimal toxicity. In the Phase I trials, a maximum tolerated dose (MTD) for STI571 has not been reached. Although responses in chronic phase patients have been durable, relapses in blast crisis patients have been common. A more rational approach to dose selection for a molecularly targeted agent would be to evaluate maximal inhibition of the target rather than using the MTD. Similarly, to evaluate mechanisms of relapse, one would want to first determine whether or not the intended target was inhibited. To achieve this goal, we have been developing tools to monitor Abl kinase inhibition. For this purpose, we have chosen three proteins that are known to be phosphorylated in CML patient samples. These proteins include Bcr-Abl itself, Crkl and STAT5. Major sites of tyrosine phosphorylation of these proteins have been mapped and phosphospecific antibodies to these specific tyrosine residues have been generated; the Abl and Crkl antisera by our laboratory and the STAT5 antibody from a commercial source. To validate these reagents, K562 cells (a human CML cell line) were incubated with 1 mM STI571 for various period of time and lysates were analyzed by immunoblotting with the phosphospecific antibodies. A dramatic decrease in the phosphorylation of Bcr-Abl is seen following a 1 hour incubation with STI571. At 48 hours, there is a complete absence of Bcr-Abl phosphorylation. The phosphospecific Crkl antisera detected a significant decrease in the phosphorylation of Crkl within 1 hour of treatment with STI571 and an 80% reduction in its phosphorylation at 48 hours. Using the phosphospecific STAT5 antibody, a complete loss of STAT5 phosphorylation was noted within 24 hours of exposure of the cells to STI571. In conclusion, we have developed specific reagents that enable us to detect kinase inhibition in a CML cell line treated with the kinase inhibitor STI571. These reagents will be tested on CML patients undergoing treatment with STI571 and should allow for direct assessment of kinase inhibition in a clinical setting. This data can be correlated with clinical responses to assist in defining optimal dosing and in determining mechanisms of relapse or resistance.
T.M. Launder, B.J. Druker, M.J. Mauro, M.E. O'Dwyer, D. Resta, R.M. Braziel Dept of Pathology; Leukemia Program, Oregon Health Sciences University, Portland, OR; Novartis Pharmaceuticals, East Hanover, NJ STI571, a specific Abl kinase inhibitor, has been shown in a phase I clinical trial to produce sustained complete hematologic responses (CHR) in chronic phase CML patients at daily doses of 300 mg or greater (Blood 94:368a, 1999). In the present study, we document the sequential hematologic and bone marrow findings in 11 chronic phase CML patients treated at OHSU as part of a multicenter study. The patients were treated with STI571 at effective doses (300-600 mg qd), and have been followed with peripheral blood (PB), marrow examinations and cytogenetics at 0, 2, 5, 8, 11 and 14 months. After 2 months of STI571 therapy, 10/11 patients had a CHR, defined as WBC < 10x109/L and PLT < 450x109/L. The remaining patient reached CHR at 5 months. In all patients, circulating myeloid precursors disappeared within 2-5 months. Basophilia was absent in 9/11 patients at 2 months and in all patients at 8 months. Cytopenias are a potential complication of STI571 therapy. At 11 months, 7 patients had mildly decreased Hct compared to pretreatment (mean decrease of 3.7%). 2/11 patients developed grade II-III neutropenia and thrombocytopenia that resolved with treatment interruption and/or dose reduction. Normalization of marrow findings tended to lag behind the PB response. Of the 10 patients showing CHR at 2 months, 7 had persistent marrow involvement with myeloid and/or megakaryocytic hyperplasia. By 8-11 months, the marrows showed no morphologic evidence of CML. STI571 produced transient myeloid and megakaryocytic hypoplasia in 7 and 4 patients, respectively and 3 patients had persistent hypoplasias at 11 months. All of the patients had at least mild reticulin fibrosis and this decreased over the course of treatment in 6 patients. 6/11 patients remain 100% Ph+ by metaphase cytogenetics analysis despite morphologic remission. The remaining 5 have had varying degrees of cytogenetic response, including one cytogenetic remission. In summary, treatment of chronic phase CML patients with STI571 at effective doses typically produces CHR within 2 months and frequently precedes bone marrow morphologic remission. The majority of patients have a mild, but clinically insignificant decrease in Hct. Neutropenia and thrombocytopenia may occur, but are easily manageable, and may represent a measure of efficacy, rather than toxicity from STI571.
M.J. Mauro, B.J. Druker, R.M. Braziel, T.M. Launder, J. Ford, M.E. O'Dwyer Leukemia Program; Department of Pathology, Oregon Health Sciences University, Portland, OR, USA; Novartis Pharmaceuticals, Basel, Switzerland Chronic myelogenous leukemia (CML) is a hematopoetic malignancy resulting from the expression of a constitutively activated BCR-ABL tyrosine kinase. STI571, an ABL tyrosine kinase inhibitor, has significant activity in CML patients, with minimal toxicity. Hematologic responses have been observed at doses 140 mg with sustained complete hematologic responses in the majority of patients treated with 300 mg. To explore possible dose response effects, time to response was measured in 19 patients treated at our institution as part of a previously reported phase I trial of STI571. Patients were treated at 11 dosage levels, ranging from 25 to 600 mg, for an average of 11 months. 5 different endpoints were analyzed at various grouped dose levels and are summarized in the table below. At doses of 25 and 85 mg of STI571, none of the five endpoints were reached. At doses of 400-600 mg of STI571, as compared to 140-350 mg, normalization of peripheral blood WBC and resolution of peripheral thrombocytosis occurred much more rapidly, at a mean of 14 and 26 days, respectively. More rapid resolution of peripheral basophilia occurred as well, at a mean of 2.3 months. In a similar fashion, bone marrow M:E ratio and cellularity changes occurred more rapidly in this higher dose cohort. These data suggest a possible dose response effect in achievement of endpoints crucial to determining response in patients with CML treated with STI571. Additionally, these observations support the use of the 400 mg dose of STI571 as has been chosen for ongoing current phase II trials.
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