Phospho-CRKL monitoring for the assessment of BCR-ABL activity in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia patients treated with nilotinib

Actual BCR-ABL kinase inhibition in vivo as determined by phospho-CRKL(pCRKL) monitoring has been recognized as a prognostic parameterin patients with chronic myelogenous leukemia treated with imatinib.We report a biomarker sub-study of the international phase Iclinical trial of nilotinib (AMN107) using the established pCRKLassay in imatinib-resistant chronic myeloid leukemia or Ph+acute lymphoblastic leukemia. A minimum dose (200 mg) requiredfor effective BCR-ABL inhibition in imatinib resistant/intolerantleukemia was determined. The pre-clinical activity profile ofnilotinib against mutant BCR-ABL was largely confirmed. Substantialdifferences between peripheral blood baseline pCRKL/CRKL ratioswere observed when comparing chronic myeloid leukemia with Ph+acute lymphoblastic leukemia. Finally, rapid BCR-ABL-reactivationshortly after starting nilotinib treatment was seen in acutelymphoblastic leukemia patients with progressive disease carryingthe P-loop mutations Y253H, E255K, or mutation T315I. Monitoringthe actual BCR-ABL inhibition in nilotinib treated patientsusing pCRKL as a surrogate is a means to establish effectivedosing and to characterize resistance mechanisms against nilotinib.

Key words: CRKL, BCR-ABL, nilotinib, resistance.

Introduction

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Introduction

Imatinib has revolutionized CML and Ph⁺ ALL therapy and is nowstandard medical treatment.^1,2 While imatinib monotherapy produceshigh response rates in early stages of CML (chronic phase; CP),more advanced CML (accelerated phase and blast crisis; AP, BC)and Ph⁺ ALL are prone to development of resistance.³ In vitroand in vivo studies have revealed that the reactivation of BCR-ABLsignaling in the presence of continued imatinib-treatment isthe major cause of resistance. In the majority of patients,reactivation can be traced to the emergence of BCR-ABL kinasedomain mutations which impair the binding of imatinib with differentiallevels of resistance induction (low, intermediate, high, complete).^4,5Measurement of BCR-ABL kinase inhibition in vivo can predictresponse to first-line treatment with imatinib.⁶ PhosphorylatedCRKL (pCRKL), a BCR-ABL adaptor protein, serves as a surrogateof BCR-ABL activity in vivo.⁷ On electrophoresis pCRKL migratesmore slowly than the unphosphorylated CRKL.⁸ Nilotinib (formerlyAMN107, Novartis Pharmaceuticals, Basel, Switzerland) is $≥$ 20-foldmore potent than imatinib in killing wild-type BCR-ABL-expressingcell lines.^9,10 Nilotinib maintains activity against 32/33 imatinib-resistantBCR-ABL mutants, but has no significant activity against theT315I mutant in vitro. Here we report the pCRKL monitoring ofpatients with imatinib resistant or intolerant CML or Ph+ ALLtreated with nilotinib in an open label dose escalation phaseI protocol.¹¹

Design and Methods

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Design and Methods

Clinical study design
The clinical study design has already been published.¹¹ Thestudy was conducted in accordance with the Declaration of Helsinkiand was approved by the institutional review board. Patientswere successively assigned to one of nine dose cohorts, rangingfrom 50 to 1,200 mg administered once daily (qd) and from 800to 1,200 mg administered as split dosage twice daily (400 bid,600 bid). Patients reported were recruited from June 2004 atFrankfurt University and represent a sub-population of the internationalstudy cohort.

Sample collection and preparation
Approximately 10 mL of EDTA-anticoagulated peripheral bloodwas collected from patients at the study site (Frankfurt) andshipped at room temperature to the laboratory (Mannheim) within24 hours. Samples were collected at baseline, and at least onceper treatment cycle (1 cycle = 28 days); in the low dose cohorts(50 and 100 mg qd) usually once a week.

Detection, quantification and mutation analysis of BCR-ABL
RNA-extraction, cDNA synthesis, qualitative and quantitativeBCR-ABL PCR were performed as previously described^12,13 Mutationanalysis was performed by denaturing high-performance liquidchromatography (D-HPLC) essentially as described by Soveriniet al.¹⁴ with slight modifications of the primers and temperatures.¹⁵The estimated proportion of mutant clone was derived from thenucleotide associated peaks as provided by sequence analysis.

Protein extraction and Western blot analysis
Western blot analysis of total leukocyte lysates to determinethe level of pCRKL was performed according to published protocols.^4,6Immunoblots were generated using the anti-CRKL antibody (C-20,Santa Cruz, Heidelberg, Germany), which detects the phosphorylatedand unphosphorylated protein fractions separated by band shift.Autoradiographs were scanned using the ChemiDoc XRS system (BioRad)to quantitate the signal intensity and to calculate the ratiopCRKL/total CRKL. Validation experiments using 10 randomly selectedpatient samples with three independent experiments revealeda coefficient of variability (CV) of 14% (3–22%).

Data analysis
Significant differences between treatment conditions were analyzedusing the t test. Data analysis was performed using the GraphPadPrism version 4.00 for Windows, GraphPad Software, San DiegoCA, and MS Excel (Seattle, WA, USA).

Results and Discussion

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Results and Discussion

Patients’ baseline characteristics
Patients’ baseline characteristics are summarized in Table 1.Kinase domain mutations were detected in 17/33 patients. Detectionof CRKL prior to treatment with nilotinib revealed > 50%pCRKL in the PB of CML patients (AP, myBC) (Figure 1A). By contrast,Ph+ ALL and lyBC patients show a significantly lower pCRKL/CRKLratio with pCRKL being <10% in the PB of 7/9 patients (p<0.01).In line with low pCRKL in ALL/lyBC is a trend towards lowerleukocyte counts and a statistically significant lower BCR-ABL/ABLratio in PB of ALL/lyBC patients (p<0.01, data not shown).The discrepancy of the pCRKL detectability in myeloid comparedwith lymphoblastic Ph+ leukemia is reminiscent of the fact thatin ALL PCR-based detection of BCR-ABL transcripts is preferablyperformed in the bone marrow (BM), whereas quantification ofBCR-ABL by RT-PCR reveals comparable results in BM and PB ofCML patients.^12,16 We therefore hypothesize that low pCRKL levelsin the PB of lyBC/Ph+ ALL patients might rather reflect differentialcompartmentalization of the leukemic clone than indicating BCR-ABLdependency of the disease in general. However, serial and parallelBM/PB analysis would be needed to prove this. From this preliminarydata we conclude that CRKL-monitoring at early time-points afterstart of treatment in the PB appears feasible in AP/BC CML.By contrast, low or undetectable pCRKL in Ph+ ALL prior to treatmentonset does not allow serial in vivo assessment of actual kinaseinhibition in PB leukocytes by Western blot analysis.

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Table 1. Patient and sample characteristics.

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Figure 1. Baseline pCRKL/CRKL ratio prior to study entry (A). Nilotinib naive patients were analyzed according to stage of disease (x-axis) based on their pCRKL/CRKL ratio (y-axis). Patients tested mutation positive by D-HPLC and direct sequencing are indicated with black symbols, mutation negative patients appear as transparent symbols. The mean pCRKL/CRKL ratio is shown as a black line. **p<0.01. Intrapatient dose escalation of nilotinib leads to mutation dependent pCRKL response (B, C). Monitoring results of 3 patients carrying BCR-ABL kinase mutations with differential sensitivity to nilotinib (M351T [left] low resistant; G250 and F359V intermediate resistant). Dose escalation is shown as increasingly darker grey shades. Serial pCRKL/CRKL ( $bullet$ ) and BCR-ABL/ABL ( ${circ}$ ) measurements are shown in percent (y-axis left) over time (study day; x-axis). The bold line indicates a best-fit analysis of pCRKL/CRKL-values. The right y-axis relates to the 10⁹/L with the corresponding data points shown as ${diamond}$ . First appearance of best response is indicated by arrows (CHR: complete hematologic response; CCR: complete cytogenetic response; RTC: reversal to chronic phase; SD: stable disease). A representative immunoblot demonstrating the CRKL-expression of the patient expressing BCR-ABL^M351T is shown. (C). Response evaluation results for leukocytes (WBC), quantitative BCR-ABL/ABL mRNA measurement, cytogenetic evaluation (CCR; complete cytogenetic response), calculated pCRKL/CRKL ratio in percent, and the estimated fraction of mutant BCR-ABL as assessed by estimating the relative size of the nucleotide peaks by sequencing. *study day 113; **day 225; #day 169; $§$ : 0 is an operational arbitrary value due to the undetectability of phosphorylated CRKL. Stage dependent CRKL-response in AP and myBC CML (D). Best pCRKL-inhibition on study is depicted as pCRKL/CRKL ratio in percent (y-axis) and compared with baseline pCRKL/CRKL ratios (prior to start of study treatment). Data points represent individual patients with the mean shown as a black line. Only patients with available baseline and on study data were selected for analysis. Hatched symbols represent patients who achieved complete hematologic response. **p<0.0001; *p<0.05.

pCRKL at progressive disease in Ph+ ALL
According to sample availability, we were able to analyze 7ALL/lyBC patients with available baseline and PD samples. Mutations,pCRKL/CRKL, BCR-ABL/ABL ratios, and estimated mutant clone fractionaccording to sequencing profile are shown in Table 2. All patientsexcept one (M244V) were recruited in cohorts with starting dailydoses $≥$ 200 mg of nilotinib. Doses at progressive disease in allcases were >200 mg. A significant increase in pCRKL/CRKLat PD was seen in all but the M244V patient. Patients carryingcomplete or high resistance mutations (T315I, E255K, Y253H)showed very short time to progression (TTP) with concomitantCRKL-phosphorylation within a 28 day time period indicatingBCR-ABL dependent progressive leukemic involvement of the PB.It is widely accepted that rapid leukemic progression on imatinibtreatment reflects selection of highly resistant clones, anddiscontinuation of inhibitor treatment has the potential toslow down disease progression.^3,17 Whether continued treatmentwith imatinib increases the oncogenic fitness of highly resistantBCR-ABL-mutants besides providing the mutant clone with a selectionadvantage is currently under investigation.^18,19

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Table 2. Clinical and molecular parameters of Ph⁺ lymphoblastic leukemias (Ph⁺ ALL and lyBC) at baseline and time of progressive disease. Patient selection is based on sample availability at baseline and progressive disease.

pCRKL-response to intrapatient dose-escalation of nilotinib
Three patients with AP-CML entering the protocol in the 50 mg(2x) or 100 mg (1x) starting dose cohorts were available fordose-response analysis. They carried resistance mutations, withone patient expressing low resistant mutant M351T, and the remainingpatients expressing the intermediate resistant mutants G250Eand F359V. All 3 of the patients presented with leukocytosisbetween 30x10⁹/L and 80x10⁹/L. BCR-ABL/ABL ratio was 100% byquantitative PCR in all 3 patients (Figure 1B). The M351T-positivepatient rapidly responded with pCRKL-suppression below 50% afterdose increase to 200 mg qd (Figure 1B,C). Complete hematologicremission (CHR) and complete cytogenetic remission (CCR) wereachieved at day 113 with low level pCRKL/CRKL still detectable.pCRKL became undetectable at day 169 after start of treatment.pCRKL kinetics of the patients with mutations G250E and F359Vare consistent with the known less sensitive in vitro responseto nilotinib.¹⁰ Interestingly, BCR-ABL/ABL reduction in all3 patients is associated with preceding pCRKL-inhibition below50%. In a recently published clinical study that prospectivelyanalyzed the pCRKL/CRKL ratio in CML patients treated with imatinib,the 50% threshold of kinase inhibition was identified as prognosticallyrelevant.⁶ Therefore, decreased clinical activity associatedwith less potent pCRKL-reduction corroborates the importanceof effective actual BCR-ABL-inhibition in patients treated withnilotinib.

CRKL inhibition may translate into disease control in some patients with advanced Ph+ leukemia
We also investigated whether inhibition of pCRKL by nilotinibis associated with clinically meaningful response. A separateanalysis for accelerated phase and BC CML was performed forpatients with detectable CRKL-phosphorylation at baseline. BaselinepCRKL/CRKL and overall best pCRKL/CRKL-inhibition on nilotinibare shown in Figure 1D. Both populations show significant pCRKL-inhibitionafter starting study treatment. In AP, 6/10 (60%) patients achievedcomplete CRKL-inhibition with 4 of them developing CHR overtime. BC patients showed complete pCRKL-inhibition in 2/6 patients(33%) with none of the patients achieving CHR over time. EffectiveCRKL-inhibition despite resistant/progressive disease was alsoobserved in patients treated with imatinib.⁴ This observationmay reflect the more complex biology of advanced disease withrecruitment of additional oncogenic signals in addition to BCR-ABL,and is therefore indicative of BCR-ABL-independent disease.²⁰Patients with BCR-ABL-independent disease are not predictedto benefit from alternative BCR-ABL-selective treatment.

To summarize, our preliminiary data support serial PB pCRKL-monitoring of patients treated with nilotinib for Ph+ myeloidleukemia as a potential means to establish effective kinaseinhibition or to detect BCR-ABL reactivation in nilotinib-resistantpatients. The limitations of this assay have been shown in advanceddisease, where effective CRKL-inhibition was not always associatedwith disease control (CML-BC), or where possible compartmentalizationto the bone marrow prevents pCRKL-analysis in the PB (Ph+ ALL).It is hoped that with the introduction of flow cytometry basedassays pCRKL monitoring becomes more feasible for clinical routine.²¹Given the increasing evidence for stem cell resistance of BCR-ABLinhibitors, detection of BCR-ABL inhibition in vivo by flowcytometry may allow us to specifically gate for the residualleukemic population within the stem cell compartment.

Acknowledgments

we would like to thank Mrs. Susanne Brendel and Mrs. Silke Willfor their expert technical assistance. We are grateful to P.Manley (Novartis) for his helpful advice on the manuscript.We thank patients who participated in the clinical trial.

Footnotes

Funding: PL is funded by the Max Eder program, Deutsche Krebshilfe,Germany.

Authorship and Disclosures

PL designed research and wrote paper; SHE, JD, TE and PE performedresearch, HK, NH, AB, and MCM analyzed data; LW and OGO treatedpatients; YS and MD designed the clinical study; RH wrote thepaper; AH designed study, wrote the paper. YS and MD are employeesof Novartis, the manufacturer of nilotinib. RH, AH and OGO receivedresearch funding by Novartis. The other authors reported nopotential conflicts of interest.

Received for publication September 3, 2007. Revision received November 26, 2007. Accepted for publication December 10, 2007.

References

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