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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-05-1366.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Hematology, University of
Leipzig, Germany; Roboscreen Gesellschaft für molekulare
Biotechnologie, Leipzig, Germany; Novartis Pharma, Basel, Switzerland;
and BMT/Leukemia Center, Oregon Health and Science University (OHSU),
Portland, OR.
Imatinib induces remissions in approximately 30% of patients with
chronic myeloid leukemia (CML) in myeloid blast crisis (M-BC). Because
most patients eventually relapse, allogeneic stem cell transplantation
(SCT) in remission offers the best chance for cure. Remission induction
with imatinib alone would seem ideal because it is less toxic than
conventional chemotherapy. Conversely, patients unlikely to respond may
benefit from combination therapy up front. To identify
prognostic factors, we studied the mRNA expression of genes related to
drug resistance and apoptosis in leukemic cells from patients with M-BC
and their in vitro sensitivity to imatinib, and analyzed the results
with other baseline factors for their impact on response. We show that
high levels of BAX, low levels of MRP-1, and a
high platelet count are independently predictive of response to
imatinib. Combined into a score, these parameters may be clinically
useful for risk-adapted patient stratification.
(Blood. 2003;101:2152-2155) Chronic myeloid leukemia (CML) in myeloid blast
crisis (M-BC) is probably incurable by chemotherapy.1,2
The results of stem cell transplantation (SCT) are poor if performed in
frank blast crisis3 but can be improved after induction of
a second chronic phase.4 Imatinib induces sustained
hematologic remissions (> 4 weeks) in approximately 30% of patients
with CML in M-BC.5 Compared with conventional
chemotherapy, imatinib is less toxic.5 Thus, remission
induction with imatinib alone prior to SCT would be desirable. By
contrast, patients unresponsive to single-agent imatinib may benefit
from combination treatment up front. To identify factors
predictive of response, we studied the in vitro sensitivity to imatinib
as well as the expression of apoptosis-related and drug
resistance-related genes by quantitative reverse
transcription-polymerase chain reaction (RT-PCR) in CD34+
cells from patients with CML in M-BC prior to imatinib therapy. In the
past, this panel of genes had been used successfully for identification
of prognostic factors in acute myeloid leukemia (AML)6 and
soft tissue sarcoma.7 Together with clinical baseline
parameters, the results were analyzed in a multivariate model for their
impact on response to imatinib.
Patients
Progenitor cell selection and in vitro sensitivity assays
Expression analysis of candidate genes Total RNA was extracted from 5 × 104 to 5 × 106 CD34+ cells with RNAeasy columns (Qiagen, Hilden, Germany) and reversely transcribed into cDNA with random hexamers.8 The expression of MRP-1, MDR-1, MDM-2, BAX, BAD, survivin, BCL-2, BCL-XL, and BCR-ABL mRNA was measured by high-throughput Taqman-PCR, normalized for GAPDH, and expressed as transcripts/amol GAPDH. Samples with low expression of GAPDH (< 0.01 amol/µL cDNA) were excluded. Details of the assays and their validation were recently published.6,7Statistical analysis The impact of clinical baseline parameters, in vitro response, and the level of expression of the various candidate genes on response to imatinib were analyzed in a logistic regression model, where the expression levels of candidate genes were introduced as categorical variables (above and below median). Only parameters with P < .1 in univariate analysis were included in the multivariate model and introduced according to the method "Wald forward." Comparison of gene expression between M-BC and normal BM was by Mann-Whitney U test. All calculations were done with the SPSS software package.
Using the criteria published by Sawyers et al,5 there
were 6 complete (13%) and 6 partial responses (13%), whereas 5 patients (11%) returned to chronic phase (overall response rate,
37%). Because the percentage of blasts was very variable,
CD34+ cells were selected to obtain a more homogenous
population of cells. As a rule, the purity of the CD34+
selections was more than 90%. Nonetheless, the expression levels of
the various mRNAs studied were highly variable (Table
1), likely reflecting the biologic
heterogeneity of M-BC. In contrast, a much more even distribution
was observed in normal bone marrow CD34+ cells. Highly
significant differences between normal cells and M-BC were observed in
the case of BCL-XL, MDR-1, MDM-2, MRP, and survivin.
For analysis of their impact on response, continuous
parameters were introduced as categorical variables (Tables
2-3).
The candidate genes were categorized according to expression above and
below median (Table 3). In univariate testing, low circulating blasts,
basophils, MRP1, and high platelets and BAX were
associated with responsive disease. In multivariate analysis, only high
platelets, high BAX, and low MRP expression
retained significance and predicted response to imatinib. Combining the
3 risk factors into a score distinguished between likely responders and
likely nonresponders (Table 4).
Bax is a proapoptotic protein and may be important for inducing an apoptotic response to imatinib. Data regarding its role in CML are limited to the notion that its level of expression is not associated with disease stage.9 Our findings in M-BC of CML are in contrast to those in AML where low levels of BAX mRNA are positively correlated with chemosensitivity.6 This discrepancy may reflect the different natures of the diseases as well as the treatments. The role of Mrp-1 in CML has also not been studied in any detail. In AML, high Mrp activity is associated with a poor response to induction chemotherapy.10 It is conceivable that, as a transmembrane transporter protein, Mrp-1 may also confer primary resistance to imatinib. All other genes tested, including the expression levels of BCR-ABL, did not influence response in multivariate analysis. There was no correlation between the in vitro response assessed by
progenitor cell assays and the response in vivo. Failure to produce
colonies was not more frequent in responders versus nonresponders
(P = .5, We also analyzed the impact of all parameters on survival in a Cox regression model. In univariate analysis, previous treatment (other than hydroxyurea) for blast crisis (P = .058), platelet counts less than 150 × 109/L (P < .0005), circulating blasts more than 50% (P = .006) were associated with shorter survival. In multivariate analysis, only low platelets retained significance (P = .003). Thus, the factors that determine the initial response to imatinib are, for the most part, distinct from those that determine survival. Ideally, this score should be confirmed in an independent series of patients. Unfortunately, because our system involves separation of CD34+ cells, no such series of sufficient size is currently available for study. If confirmed, the score may allow identifying those patients who are likely to respond to imatinib alone. In the context of a subsequent SCT, where outcome is much better for patients receiving transplants in remission,4,11 this would be particularly advantageous; remission could be induced without the toxicity associated with conventional chemotherapy, which might in turn improve performance status at the time of SCT. By contrast, patients unlikely to respond to imatinib alone may benefit by combining imatinib with conventional cytotoxic drugs up front. In vitro studies indicate that this approach may be effective.12
Submitted May 10, 2002; accepted October 20, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-05-1366.
Supported by a research grant from Novartis Pharmaceuticals, Basel, Switzerland.
I.v.H. is an employee of Novartis Pharma, Basel, Switzerland.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael Deininger, OHSU, BMT/Leukemia Center, 3181 SW Sam Jackson Park Rd, Portland, OR 97239; e-mail: deininge{at}ohsu.edu.
1.
Wadhwa J, Szydlo RM, Apperley JF, et al.
Factors affecting duration of survival after onset of blastic transformation of chronic myeloid leukemia.
Blood.
2002;99:2304-2309 2. Sacchi S, Kantarjian HM, O'Brien S, et al. Chronic myelogenous leukemia in nonlymphoid blastic phase: analysis of the results of first salvage therapy with three different treatment approaches for 162 patients. Cancer. 1999;86:2632-2641[CrossRef][Medline] [Order article via Infotrieve]. 3. Gratwohl A, Hermans J, Goldman JM, et al. Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow transplantation. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Lancet. 1998;352:1087-1092[CrossRef][Medline] [Order article via Infotrieve]. 4. Visani G, Rosti G, Bandini G, et al. Second chronic phase before transplantation is crucial for improving survival of blastic phase chronic myeloid leukaemia. Br J Haematol. 2000;109:722-728[CrossRef][Medline] [Order article via Infotrieve].
5.
Sawyers CL, Hochhaus A, Feldman E, et al.
Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study.
Blood.
2002;99:3530-3539 6. Kohler T, Schill C, Deininger MW, et al. High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML). Leukemia. 2002;16:22-29[CrossRef][Medline] [Order article via Infotrieve]. 7. Wurl P, Kappler M, Meye A, et al. Co-expression of survivin and TERT and risk of tumour-related death in patients with soft-tissue sarcoma. Lancet. 2002;359:943-945[CrossRef][Medline] [Order article via Infotrieve].
8.
Deininger M, Goldman JM, Lydon NB, Melo JV.
The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL positive cells.
Blood.
1997;90:3691-3698 9. Ravandi F, Kantarjian HM, Talpaz M, et al. Expression of apoptosis proteins in chronic myelogenous leukemia: associations and significance. Cancer. 2001;91:1964-1972[CrossRef][Medline] [Order article via Infotrieve]. 10. Laupeze B, Amiot L, Drenou B, et al. High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival. Br J Haematol. 2002;116:834-838[CrossRef][Medline] [Order article via Infotrieve]. 11. Deininger MWN, Schäfer K, O'Brien S, et al. STI571 prior to allogeneic stem cell transplantation (SCT): a retrospective analysis by the European blood and marrow transplantation group (EBMT) and the international STI571 investigators [abstract]. Hematol J. 2001;1:96.
12.
Thiesing JT, Ohno-Jones S, Kolibaba KS, Druker BJ.
Efficacy of STI571, an abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against bcr-abl-positive cells.
Blood.
2000;96:3195-3199
© 2003 by The American Society of Hematology.
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  M. W. Deininger Milestones and Monitoring in Patients with CML Treated with Imatinib Hematology, January 1, 2008; 2008(1): 419 - 426. [Abstract] [Full Text] [PDF]
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 S. H. Kaufmann Imatinib spells BAD news for Bcr/abl-positive leukemias PNAS, October 3, 2006; 103(40): 14651 - 14652. [Full Text] [PDF]
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 N. Theou, S. Gil, A. Devocelle, C. Julie, A. Lavergne-Slove, A. Beauchet, P. Callard, R. Farinotti, A. Le Cesne, A. Lemoine, et al. Multidrug Resistance Proteins in Gastrointestinal Stromal Tumors: Site-Dependent Expression and Initial Response to Imatinib Clin. Cancer Res., November 1, 2005; 11(21): 7593 - 7598. [Abstract] [Full Text] [PDF]
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 M. Deininger, E. Buchdunger, and B. J. Druker The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood, April 1, 2005; 105(7): 2640 - 2653. [Abstract] [Full Text] [PDF]
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P. T. Ferrao, M. J. Frost, S.-P. Siah, and L. K. Ashman Overexpression of P-glycoprotein in K562 cells does not confer resistance to the growth inhibitory effects of imatinib (STI571) in vitro Blood, December 15, 2003; 102(13): 4499 - 4503. [Abstract] [Full Text] [PDF]
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 M. W. N. Deininger and B. J. Druker Specific Targeted Therapy of Chronic Myelogenous Leukemia with Imatinib Pharmacol. Rev., September 1, 2003; 55(3): 401 - 423. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2). Because the cytokine mix used
supports not only the growth of blasts but also of mature colonies, it
is possible that the assay predominantly favored the proliferation of
progenitor cells that did not belong to the blastic cell clone and may
as such not reflect the clinical situation.