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Measurement of In Vivo BCR-ABL Kinase Inhibition to Monitor Imatinib-Induced Target Blockade and Predict Response in Chronic Myeloid Leukemia

Deborah White, Verity Saunders, Andrew Grigg, Chris Arthur, Robin Filshie, Michael F. Leahy, Kevin Lynch, L. Bik To, Timothy Hughes

From the Division of Hematology, Institute of Medical and Veterinary Science & Hanson Institute; The University of Adelaide; and Royal Adelaide Hospital, Adelaide, South Australia; Royal Melbourne Hospital, Melbourne; Royal North Shore Hospital, Sydney; St Vincent's Hospital, Melbourne; Fremantle Hospital, Perth; and Novartis Pharmaceuticals Australia Pty Ltd, Sydney, Australia

Address reprint requests to Deborah White, Division of Hematology, Institute of Medical and Veterinary Science & Hanson Institute, PO Box 14, Rundle Mall Post Office, Adelaide, Australia 5001; e-mail: deb.white{at}imvs.sa.gov.au

	ABSTRACT

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Purpose Intrinsic sensitivity to imatinib, based on measurement of inhibitory concentration 50% for imatinib, is variable in untreated patients with chronic myeloid leukemia (CML). This suggests that patient-tailored dosing may be more rational than a fixed dose for all. Dose optimization potentially could be based on accurate measurement of the level of BCR-ABL kinase inhibition achieved in vivo.

Patients and Methods In vivo kinase inhibition was measured by calculating the reduction in protein (p) -Crkl level in mononuclear blood cells taken from 49 CML patients at weekly intervals after imatinib therapy was commenced.

Results Greater than 50% inhibition (> 50% reduction in p-Crkl from baseline) was achieved by 21% of patients by days 7 to 14 (and maintained in all patients on days 21 to 28) and an additional 24% of patients achieved more than 50% inhibition by days 21 to 28. Thus, overall 45% of patients achieved more than 50% inhibition. All of these patients achieved major molecular responses by 24 months compared with 56% of the patients who failed to achieve 50% kinase inhibition (P < .001). Patients with less than 50% kinase inhibition were also more likely to have suboptimal responses.

Conclusion In vivo BCR-ABL kinase inhibition can be assessed in the first month of imatinib therapy and may provide a valuable guide to optimization of dosage. The extent of BCR-ABL kinase inhibition is an excellent predictor of cytogenetic and molecular response. These observations suggest that dose adjustment based on in vivo measurements of drug-induced target inhibition could be applied in settings beyond imatinib and may be a more effective approach than using one dose for all patients in targeted anticancer therapy.

	INTRODUCTION

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The cytogenetic hallmark of chronic myeloid leukemia (CML) is the Philadelphia chromosome (Ph), which results in the formation of the BCR-ABL fusion gene encoding a 210-kd protein.^1-3 This fusion protein is an active tyrosine kinase, critical to the pathogenesis of chronic myeloid leukemia (CML).^4-6 Imatinib (Gleevec; Novartis Pharmaceuticals, East Hanover, NJ) is an ABL tyrosine kinase inhibitor that has become the paradigm for molecularly targeted anticancer therapies.⁷ Clinical trials have demonstrated the efficacy of imatinib in chronic-phase CML.^8,9 Five-year International Randomised Study of Interferon versus STI571 (IRIS) data indicates 89% of patients surviving to this time point.^10,11 At the molecular level, significant reductions in the level of BCR-ABL transcript, measured by quantitative real-time polymerase chain reaction have been observed.¹² One hundred percent of patients achieving a 3 log or greater reduction in the level of BCR-ABL from standardized baseline by 12 months of imatinib therapy (major molecular response [MMR]) remain transformation free to 5 years. This compares with 95% of patients who achieve complete cytogenetic response but fail to achieve MMR by 12 months (P = .007),¹⁰ and indicates that achievement of MMR is a significant landmark in the longer term patient response. Although these outcomes demonstrate excellent overall outcomes for imatinib-treated CML patients, up to 20% of patients have suboptimal response and a significantly higher risk of disease progression.¹³ In addition, 5% to 10% of patients develop imatinib resistance, commonly due to mutations within the ABL kinase domain,^14,15 which prevent or reduce the efficacy of imatinib binding.

The striking success of imatinib is even more remarkable given that patients are treated with a one-dose-for-all strategy. Pharmacokinetic studies indicate that dose is the strongest predictor of overall drug exposure and there is no necessity for imatinib dosing on a milligram-per-kilogram basis.^16,17 However, the poor response observed in a minority of patients indicates a role for dose escalation in selected patients. This strategy necessitates the development of reliable assays to predict, early in the treatment course, patients who may benefit from dose escalation. A limited role has been demonstrated for classical prognostic indicators such as the Sokal score¹⁸ in imatinib-treated patients. Response indicators, such as achievement of MMR by 12 months, are better predictors of long-term response,¹⁹ which attenuate any subsequent predictive value of the Sokal score. Given that imatinib is a rationally designed therapy targeting the kinase activity of BCR-ABL, a logical approach to dose optimization is the development of assays that measure the degree of target inhibition achieved, allowing assessment of the effect of imatinib in vivo. We²⁰ and others²¹ have shown previously that the inhibitory concentration 50% for imatinib (IC50^imatinib), based on the in vitro sensitivity of blood cells to imatinib, as measured by the level of phosphorylation of the adaptor phosphorylated Crkl (p-Crkl),^22,23 is predictive of molecular response. Crkl is an immediate downstream substrate for BCR-ABL; phosphorylation of Crkl occurs as a direct consequence of BCR-ABL expression, with levels of p-Crkl correlating with kinase activity of BCR-ABL protein.²³

In this study we investigated the biologic relevance and predictive value of the in vivo level of kinase inhibition achieved in patients during the first 28 days of imatinib therapy. This in vivo response reflects a summation of the effects of intrinsic sensitivity (IC50^imatinib) to imatinib-induced kinase inhibition, actual dose received, and pharmacokinetic factors such as GI absorption and hepatic metabolism. We hypothesized that the actual level of ABL kinase inhibition achieved would correlate with clinical outcome and hence be the most rational way to assess the adequacy of therapy and the potential value of dose escalation. We report the measured level of in vivo kinase inhibition is a highly significant predictor of response.

	PATIENTS AND METHODS

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Patient Population
Patients in this study were enrolled onto the Trial of Initial Intensified Imatinib Therapy and Sequential Dose-Escalation (TIDEL), a phase II study conducted in 103 adult patients with newly diagnosed CML. All patients enrolled were within 9 months of diagnosis, with no prior history of imatinib or interferon therapy. This study mandated commencement of imatinib at 600 mg/d, with dose increase to 800 mg/d if a patient failed to achieve predetermined criteria.²⁰ No patient in this study received an escalated dose before 12 months.

In a subpopulation of patients consenting to correlative science studies (Appendix Table A1, online only), blood was obtained immediately before the commencement of imatinib therapy and at 7-day intervals during imatinib therapy to day 28. All blood was collected with informed consent in accordance with the Declaration of Helsinki.

Preparation of Protein Lysates, Western Blot Analysis, and Calculation of Percentage Kinase Inhibition
Mononuclear cells were isolated from blood using Lymphoprep (Axis Shield, Oslo, Norway). A total of 2 x10⁶ mononuclear cells were lysed in Laemmli's buffer²⁴ by boiling for 12 minutes. Lysates were clarified by microcentrifugation for 5 minutes at 15,000 relative centrifugal force and stored at –20°C until Western blot was performed.

Sequential samples from the same patient were run on the same gel to minimize interassay variability. Twenty microliters of protein lysate (corresponding to 2 x 10⁶ cells) was resolved on an sodium dodecyl sulfate/10% polyacrylamide gel. Protein was electrophoretically transferred to a polyvinylidene difluoride (Amersham, Piscataway, NJ) membrane and probed with anti-Crkl antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were detected with enhanced chemifluorescence substrate (Amersham Pharmacia) and analyzed by the Fluor Imager (Molecular Dynamics, Sunnyvale, CA). Signals were quantified using Image Quant software (Molecular Dynamics).

The percentage p-Crkl (%p-Crkl) was determined using the following formula: %p-Crkl = (pCrkl/pCrkl + Crkl) x 100%

The percentage kinase inhibition at each time point was calculated by subtracting the %p-Crkl at a designated time point in each patient from the %p-Crkl at baseline of that patient. This was then converted to a percentage reduction in p-Crkl, using the following formula: percentage kinase inhibition = [(%p-Crkl at baseline – %p-Crkl at designated time point)/%p-Crkl at baseline for the individual patient] x 100%.

All data were analyzed independently at least twice, and if there was a lack of concordance, a third analysis was performed. A negative control (BCR-ABL–negative cell line) and a positive control (BCR-ABL–positive cell line) were included in each assay. The reliability and reproducibility of the assay was verified by performing three independent Western blots on 100 CML patients referred to our center. Correlations between assays ranged from 0.834 to 0.894 and were significantly different from zero (P < .0001).

IC50^imatinib determinations were performed as described previously.²⁰

Statistics
All statistical analyses were performed using SigmaStat Software (Systat Inc, San Jose, CA). Kaplan-Meier curves were constructed to estimate probability and log-rank survival analysis provided comparative statistics. Correlation was assessed using Spearman rank or Pearson product moment analyses. Repeated-measures analysis, t test, and analysis of variance were used as appropriate.

	RESULTS

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Examination of the Degree of Kinase Inhibition Achieved During the First 28 Days of Imatinib Therapy
The samples from a total of 49 patients, all of whom had baseline studies performed, were analyzed in this study. The median and range of %p-Crkl are shown in Appendix Table A2 (online only).

The degree of kinase inhibition achieved in the first 2 weeks of imatinib treatment was compared with that achieved at the third and fourth week. The kinase inhibition achieved at both days 7 and 14 correlated significantly with the degree of kinase inhibition achieved at days 21 and 28 (P < .001). The four patients who achieved more than 50% kinase inhibition on day 7 all achieved at least this level of inhibition at later time points. Similarly, those patients who achieved more than 50% kinase inhibition by day 14 (n = 6) and day 21 (n = 3) all maintained this level or a greater level of kinase inhibition to day 28. Patients who did not achieve more than 50% kinase inhibition by day 7 (n = 45) had a 39% incidence of achieving this level of response by day 28. These data make it highly probable that the observed interpatient variability is due to real differences in the level of kinase inhibition achieved.

Significance and Predictive Value for Achievement of MMR: 50% Reduction in p-Crkl
MMR was achieved in 51% of patients by 12 months and in 75% of patients by 24 months. Patients who achieved MMR by 24 months had consistently higher levels of kinase inhibition at days 7, 14, 21, and 28. This difference was statistically significant at days 7 and 21 (Fig 1A) However there was no such association observed between the white cell count (Fig 1B), or the neutrophil-to-lymphocyte ratio assessed at the same time points (Fig 1C).

View larger version (12K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Patients were grouped according to their achievement of major molecular response MMR or not by 24 months. These groups were then examined relative to the following parameters measured to day 28. (A) The percentage kinase inhibition, (B) change in white cell count, and (C) change in neutrophil-to-lymphocyte ratio.

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Patient's maximum level of in vivo kinase inhibition during the first 28 days was first ranked and then divided into quartiles. The average log reduction in BCR-ABL as measured by quantitative real-time polymerase chain reaction was then assessed based on these quartile groups. Statistical significance was determined using one-way repeated-measures analysis of variance.

View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Patients were divided into three groups: those failing to achieve 50% kinase inhibition by day 28, those achieving 50% kinase inhibition by day 14, and those achieving 50% kinase inhibition on days 21 to 28. Groups were compared to the log reduction in BCR-ABL over time.

In Vivo Kinase Inhibition and the Prediction of Suboptimal Response
All patients in this study with more than 50% kinase inhibition (n = 22) achieved a 1 log reduction by 6 months, compared with 85% (23 of 27) of patients with less than 50% kinase inhibition (P = .005). The respective figures for the achievement of a 2 log reduction by 12 months were 100% and 67% (P = .002). Ninety-five percent of patients with more than 50% kinase inhibition achieve MMR by 18 months, compared with 52% of patients with less than 50% kinase inhibition (P = .001). These data indicate that in vivo kinase inhibition as measured in the first 4 weeks of therapy is a predictor of suboptimal clinical response, and that failure to achieve more than 50% kinase inhibition in the first 28 days increases the probability of suboptimal response²⁵ (Table 1).

Other Early Response Predictors and In Vivo Kinase Activity
Combination of IC50^imatinib and in vivo kinase inhibition. In this study IC50^imatinib assays (a measure of intrinsic sensitivity of the kinase to imatinib) were performed on only 28 patients. There was good correlation between the IC50^imatinib and the percentage kinase inhibition by day 28 (r = –0.403; P = .019; n = 28; Appendix Fig A1, online only), indicating that a higher IC50^imatinib predicts for poorer in vivo kinase activity. An IC50^imatinib of less than 0.85 µM seems to be essential but is not sufficient to achieve more than 50% kinase inhibition (Fig 4). By 24 months, 13 of 13 low IC50^imatinib patients with more than 50% kinase inhibition achieved MMR, compared with only two (22%) of nine low IC50^imatinib patients with less than 50% kinase inhibition (P = .001). There were insufficient patients with a high IC50^imatinib and 50% kinase inhibition (n = 4) for statistical analysis.

View larger version (8K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 4. Patients were first divided into those who achieved more than 50% kinase inhibition, and those who failed to achieve 50% kinase inhibition by day 28. This graph demonstrates the inhibitory concentration 50% for imatinib (IC50imatinib) plotted against the two groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
In this study we demonstrate that the in vivo level of BCR-ABL kinase inhibition can be measured during the first 28 days of imatinib therapy, and identify considerable interpatient variability. Significantly, this variability delineates a group of patients with more than 50% in vivo kinase inhibition who achieve rapid molecular responses and a group who fail to achieve this level of kinase inhibition and respond less well. Early identification of these patients provides an opportunity to intervene, either by increasing imatinib dose to inhibit more strongly the BCR-ABL kinase, or by considering substitution of imatinib with a more potent ABL kinase inhibitor. The observation that all patients achieving more than 50% inhibition of ABL kinase activity achieve MMR suggests the level of kinase inhibition is the main determinant of the extent of molecular response achieved, and that failure to achieve MMR is due predominantly to inadequate kinase inhibition.

We demonstrate that the degree of in vivo kinase inhibition achieved in the first month of therapy is a superior predictor of long-term molecular response than previously defined criteria such as Sokal score, IC50^imatinib, and 3-month molecular response. Only patients with low IC50^imatinib achieve good in vivo kinase inhibition; however the converse is not true. This suggests that although a low IC50^imatinib is required to achieve more than 50% kinase inhibition in vivo, other factors, such as the actual imatinib dose received and pharmacokinetic variables, also contribute.

Another potentially important strategy to enable imatinib dose optimization is to monitor its level in the blood. Plasma imatinib levels were not assessed in this study. However, although blood levels of imatinib will take into account most of the critical variables determining target inhibition, they will not account for variation in the efficiency of intracellular transport, which we have shown is highly variable in CML patients. Assessing the relative value of IC50^imatinib, OCT-1 functional assays, imatinib drug levels, and in vivo kinase inhibition in prospective studies will clarify the role of each of these assays in dose optimization.

Dividing patients into two cohorts according to maximal in vivo kinase inhibition to analyze outcome may provide an impression that there are two distinct groups of responders: good and poor. In fact, if the patients are divided into quartiles according to their maximal level of kinase inhibition achieved, it appears there is a steady improvement in molecular response as the level of kinase inhibition increases (Fig 2). This is consistent with the concept that BCR-ABL kinase inhibition remains partial in most patients and that the optimal level of kinase inhibition is not often achieved with imatinib therapy.

Current treatment recommendations for imatinib therapy in CML patients are based on a fixed-dosing strategy derived from pharmacokinetic analyses in early clinical studies. This strategy was based on data indicating no value in adjusting standard recommended dosage (400 mg) based on age, weight, ethnicity, sex, or comedications.¹⁷ Phase II studies validated the use of higher doses (600 mg) in patients with advanced disease.^29-31 There is preliminary evidence that an even higher dose (800 mg) results in more profound responses at earlier time points,³² a finding currently being examined in prospective randomized studies. No pretherapy or early treatment phase screening strategies are currently used to select optimal dose for individual patients in chronic phase.

We present a novel assay for determining the level of ABL kinase inhibition achieved in vivo in imatinib-treated CML patients. We demonstrate kinase inhibition is highly variable between patients, and that the level of kinase inhibition achieved is highly predictive of cytogenetic and molecular response. If the predictive value of in vivo BCR-ABL kinase inhibition assays are confirmed prospectively, this raises the possibility that imatinib dose could be titrated against in vivo measures of kinase inhibition to achieve optimal target inhibition. Patients with suboptimal kinase inhibition may benefit from higher doses, and patients with complete kinase inhibition and significant toxicity may be able to receive a reduced dose safely as long as kinase inhibition remains adequate. This probably will represent a more effective strategy than a fixed dose for all patients and may lead to better outcomes for patients with poor ABL kinase inhibition. The value of individualized dosing with other small molecule inhibitors based on measures of target inhibition requires formal assessment. Although assays of target inhibition in solid tumors will not always be as convenient as a blood test, titration of target inhibition is a promising development in the search for targeted anticancer therapy that is both safe and effective.

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 

View larger version (8K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. Dot plot showing the correlation between the inhibitory concentration 50% for imatinib (IC50imatinib) and the maximal percentage kinase inhibition (reduction in in vivo percentage phosphorylated-Crkl) achieved by day 28 of imatinib therapy.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

Employment: Kevin Lynch, Novartis Pharmaceuticals Leadership: N/A Consultant: Andrew Grigg, Novartis Pharmaceuticals; Chris Arthur, Novartis Pharmaceuticals; Timothy Hughes, Novartis Pharmaceuticals Stock: Kevin Lynch, Novartis Pharmaceuticals Honoraria: Deborah White, Novartis Pharmaceuticals; Andrew Grigg, Novartis Pharmaceuticals; Chris Arthur, Novartis Pharmaceuticals; Timothy Hughes, Novartis Pharmaceuticals Research Funds: Deborah White, Novartis Pharmaceuticals; Andrew Grigg, Novartis Pharmaceuticals; Robin Filshie, Novartis Pharmaceuticals; Michael F. Leahy, Novartis Pharmaceuticals; L. Bik To, Novartis Pharmaceuticals; Timothy Hughes, Novartis Pharmaceuticals Testimony: N/A Other: N/A

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Conception and design: Deborah White, Andrew Grigg, Kevin Lynch, Timothy Hughes

Administrative support: Deborah White, Timothy Hughes

Provision of study materials or patients: Andrew Grigg, Chris Arthur, Robin Filshie, Michael F. Leahy, Timothy Hughes

Collection and assembly of data: Deborah White, Verity Saunders

Data analysis and interpretation: Deborah White, Andrew Grigg, Timothy Hughes

Manuscript writing: Deborah White, L. Bik To, Timothy Hughes

Final approval of manuscript: Deborah White, Verity Saunders, Andrew Grigg, Chris Arthur, Robin Filshie, Michael F. Leahy, Kevin Lynch, L. Bik To, Timothy Hughes

	ACKNOWLEDGMENTS

 
We thank John Reynolds, PhD, for his significant contributions to this study, and Bruce Lyons, PhD, Michael Copeman, MD, Susan Branford, PhD, Rebecca Lawrence, Chani Field, and Rachel Koelmeyer for their support and contributions.

	NOTES

 
Supported in part by a grant from the Cancer Council of Australia, with additional support from Novartis Australia, the Australasian Leukemia Lymphoma Group, Novartis, and Amgen Pharmaceuticals.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted November 13, 2006; accepted July 3, 2007.

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