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Phase I Pharmacokinetic and Pharmacodynamic Study of Temozolomide in Pediatric Patients With Refractory or Recurrent Leukemia: A Children's Oncology Group Study

Terzah M. Horton, Patrick A. Thompson, Stacey L. Berg, Peter C. Adamson, Ashish M. Ingle, M. Eileen Dolan, Shannon M. Delaney, Madhuri Hedge, Heidi L. Weiss, Meng-Fen Wu, Susan M. Blaney

From the Texas Children's Cancer Center/Baylor College of Medicine; Diagnostic Sequencing Laboratory, Medical Genetics Laboratories, and Breast Center, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX; Children's Hospital of Philadelphia, Philadelphia, PA; Children's Oncology Group Operations Center, Arcadia, CA; and Section of Hematology-Oncology, Cancer Research Center and Committee on Clinical Pharmacology, The University of Chicago, Chicago, IL

Address reprint requests to Terzah M. Horton, MD, PhD, 6621 Fannin, MC 3-3320, Baylor College of Medicine, Houston, TX 77030; e-mail: tmhorton{at}txccc.org

	ABSTRACT

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Purpose To determine the tolerability, pharmacokinetics, and mechanisms of temozolomide resistance in children with relapsed or refractory leukemia.

Patients and Methods Cohorts of three to six patients received 200 or 260 mg/m²/d of temozolomide by mouth daily for 5 days every 28 days. Toxicities, clinical response, and pharmacokinetics were evaluated. Pretreatment leukemia cell O⁶-methylguanine–DNA methyltransferase (MGMT) activity, tumor and plasma MGMT promoter methylation, and microsatellite instability (MSI) were examined in 14 of 16 study patients and in tissue bank samples from children with acute leukemia not treated with temozolomide (MGMT, n = 67; MSI, n = 65).

Results Sixteen patients (nine female, seven male; acute lymphoblastic leukemia [ALL], n = 8; acute myeloid leukemia [AML], n = 8), median age 11 years (range, 1 to 19 years), received either 200 mg/m²/d (nine enrolled, three assessable for toxicity) or 260 mg/m²/d (seven enrolled, three assessable for toxicity) of temozolomide. Temozolomide was well tolerated and no dose-limiting toxicities occurred. The mean clearance of temozolomide was 107 mL/min/m², with a volume of distribution of 20 L/m² and half-life of 109 minutes. MGMT activity in leukemia cells was quite variable and was highest in patients with relapsed ALL. Only one patient had MSI. Two patients had a partial response. Both of these patients had no detectable MGMT activity; both also had methylated MGMT promoters and were MSI stable.

Conclusion Temozolomide was well tolerated at doses as high as 260 mg/m²/d for 5 days in children with relapsed or refractory leukemia. Increased MGMT activity may account for the temozolomide resistance in children with relapsed leukemia. Leukemia cell MGMT activity was higher in pediatric ALL than AML (P < .0001).

	INTRODUCTION

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Although cure rates for children with newly diagnosed leukemia approach 80% to 90%, the prognosis for children with relapsed leukemia remains poor and novel treatment approaches are needed. Temozolomide, an imidazole tetrazinone approved for the treatment of high-grade glioma,^1,2 inhibits cell growth in leukemia cell lines and leukemia xenografts.^3,4 In a phase I study of temozolomide in adults with leukemia, two patients achieved a complete response (CR or CR without platelet recovery [CRp]) with durable remissions (8 to 14 months). The dose-limiting toxicity (DLT) was prolonged bone marrow aplasia and the maximum tolerated dose (MTD) was 200 mg/m²/d for 7 days.⁵

Temozolomide resistance correlates with activity of the DNA repair protein O⁶-methylguanine–DNA methyltransferase (MGMT).^6-8 Increased MGMT activity is associated with inferior outcomes in adult and pediatric malignant gliomas.^9,10 Several in vitro studies have shown that increased MGMT activity can contribute to temozolomide resistance in leukemia^11,12; however, correlations between patient leukemia cell MGMT activity and temozolomide resistance have not been examined directly.

Hypermethylation of cytosine-phosphate guanine (CpG) islands near gene promoter regions results in transcriptional inactivation of many genes.¹³ MGMT promoter methylation is associated with both decreased MGMT activity¹⁴ and increased response to chemotherapy in both CNS tumors^9,15 and diffuse large B-cell lymphomas.¹⁶ However some patients with leukemia have evidence of MGMT promoter hypermethylation^17,18 and it is not known whether MGMT methylation correlates with temozolomide response.

Although increased MGMT activity is associated with temozolomide resistance in AML patients and leukemia cell lines,^3,19 some leukemia cells remain resistant to temozolomide despite MGMT inhibition.³ Temozolomide resistance in these tumor cells results from mismatch repair (MRS) mutations,^20-23 which frequently are associated with microsatellite instability (MSI).²³ The frequency of MSI and its contribution to temozolomide resistance in pediatric leukemia have not been examined previously.

In this study we examined the efficacy, toxicities, and pharmacokinetics of oral temozolomide administered daily for 5 days at two dose levels, 200 and 260 mg/m²/d. In addition, we evaluated MGMT enzymatic activity, MGMT promoter methylation, and MSI in leukemia blasts from patient samples.

	PATIENTS AND METHODS

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Patient Eligibility
Patients between the ages of 1 and 21 years (inclusive) with refractory ALL or AML (> 25% blasts) were eligible for this trial. Other eligibility criteria included a Karnofsky or Lanksy performance score 50; recovery from the acute toxic effects of prior chemotherapy, radiotherapy, or immunotherapy with a minimum elapsed period of at least 7 days since the last dose of corticosteroids or hematopoietic growth factors; at least 3 months since the last stem-cell transplantation; and at least 3 months since prior craniospinal radiation, pelvic radiation, or total-body irradiation, at least 2 weeks since local palliative radiation, and at least 6 weeks since other substantial radiation. Patients could have no evidence of active graft-versus-host disease. Patients were required to have adequate renal function (serum creatinine below the upper limits of normal for age or a glomerular filtration rate 70 mL/min/1.73 m²), adequate liver function (serum bilirubin 1.5 mg/dL, ALT 5x the institutional upper limit of normal for age, and albumin 2 g/dL), a platelet count 20,000/µL, and a hemoglobin level 8 g/dL. Transfusion support was allowed if necessary to meet hematologic parameters. Study exclusion criteria included CNS leukemia, pregnancy, or lactation in women of child-bearing age, uncontrolled infection, hyperleukocytosis (WBC > 100,000 cells/µL), receipt of concomitant enzyme-inducing anticonvulsants, or concomitant use of other experimental agents. Hydroxyurea was permitted up to 24 hours before the start of therapy if needed for cytoreduction. Institutional review board (IRB) approval and informed consent from the patient or their parent(s), and assent, as appropriate, were obtained in accordance with federal and institutional policies.

Dosage and Drug Administration
Temozolomide capsules (Schering-Plough Research Institute, Kenilworth, NJ) were administered orally 1 hour before or 2 hours after meals. Capsules could be opened and the contents resuspended in apple juice or applesauce for children unable to swallow capsules.

Trial Design
The starting dose of temozolomide was 200 mg/m²/dose for 5 days (70% of the adult MTD).⁵ Subsequent planned dose escalations were in increments of 30%. Courses were repeated every 28 days if there was no evidence of progressive disease, the platelet count was 20,000/µL, the hemoglobin level was 8 g/dL (with transfusion support), and any other treatment-related adverse events were grade 1. Doses were not held or omitted for hematologic toxicity or transfusions. A minimum of three patients assessable for DLT were entered at each dose level. Dose levels were expanded to six patients if one patient experienced a DLT during the first course of therapy.

Toxicity Assessment
Adverse events were graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (version 3.0). Nonhematologic DLT was defined as any grade 3 or 4 adverse event attributable to temozolomide with the specific exclusion of grade 3 nausea or vomiting, grade 3 hepatic aminotransferase (AST and/or ALT) elevation returning to grade 1 before the next treatment course, and grade 3 fever or infection.

Response Assessment
CR was defined as the attainment of an M1 bone marrow (< 5% blasts and adequate marrow cellularity) with no evidence of circulating blasts or extramedullary disease. Partial response (PR) was defined as the complete disappearance of circulating blasts and achievement of M2 marrow status ( 5% to < 25% blast cells with adequate cellularity). Progressive disease (PD) was defined as an increase of at least 25% in the number of circulating leukemia cells, development of extramedullary disease, or other laboratory or clinical evidence of PD. Stable disease was defined as failing to fulfill the criteria for a CR, PR, or PD.

Pharmacokinetic Studies
Blood samples (2 mL) were collected in heparinized tubes before and at 0.5, 1, 2, 3, 4, 6, and 8 hours after the first temozolomide dose. Plasma was separated and frozen at –80°C until analysis. Plasma temozolomide concentrations were measured using a modification of a previously described high-performance liquid chromatography assay.²⁴ In brief, plasma treated with phosphoric acid (20 µg/mL) underwent solid-phase extraction using 30 mg/mL StrataX reverse-phase columns (Phenomenex, Torrance, CA). Plasma was eluted with acetonitrile and evaporated to dryness under nitrogen at 37°C. Samples were reconstituted in 1% phosphoric acid and injected onto a Luna-2 C18, 3 µm, 4.6 mm x 250 mm column with a C18, 3 µm, 2 mm x 3 mm guard column (Phenomenex), and eluted with a mobile phase of 0.5% acetic acid/methanol (88/12, vol/vol). Peaks were monitored on a Waters 996 photodiode array detector at 330 nm (Waters Associates, Milford, MA). Recovery of temozolomide was 79% ± 2% and the intra- and interassay precisions were 1.7% and 3.9%, respectively. The lower limit of quantitation for temozolomide was 0.012 µg/mL.

Temozolomide pharmacokinetic analyses were performed using both compartmental and noncompartmental methods. Time to maximum concentration (T_max), peak plasma concentration (C_max), area under the concentration-time curve (AUC), and apparent volume of distribution at steady state (V_d-ss/F) were calculated using noncompartmental methods.²⁵ One- and two-compartment models were fit to the concentration-time data using ADAPT II (Biomedical Simulations Resources, University of Southern California, Los Angeles, CA) with maximum likelihood estimation.²⁶ Akaike's information criterion was used to select the best model fit.²⁷ Individual patient clearance (Cl/F), the absorption half-life, and the elimination half-life for each phase were estimated from the model.²⁵

Tumor Cell Isolation
Pretreatment blood and bone marrow leukemia cells were collected, shipped, and processed as described previously.²⁸ After plasma removal, samples with less than 90% tumor cells were sorted to more than 90% purity using a DakoMation cell sorter (DAKO, Glostrup, Denmark).²⁸ Viability of each patient sample was 90% as determined by Trypan blue exclusion. Plasma, tumor cells, and normal lymphocytes (when available) were frozen in RPMI-1640 medium supplemented with 20% bovine growth serum and 10% dimethylsulfoxide.²⁸ An additional 67 previously banked leukemia specimens were obtained from the Texas Children's Cancer Center tissue bank using an IRB-approved protocol.

MGMT Activity
MGMT repair activity in peripheral-blood mononuclear cell or bone marrow aspirate tumor cell lysates was determined by the removal of O⁶-[³H] methylguanine from a [³H]-methylated DNA substrate and quantitated as femtomoles O⁶-[³H] methylguanine/mg total protein lysate.²⁹ All samples contained more than 90% leukemia cells except patients 5 (44% blasts), 12 (75% blasts), and 15 (50% blasts). Elevated MGMT activity and low MGMT activity were defined as more than 2 standard deviations from the mean MGMT activity from four normal volunteers.

MGMT Promoter Methylation
MGMT promoter DNA methylation patterns were determined using either tumor cell DNA or free DNA isolated from patient plasma using methylation-specific polymerase chain reaction, as described.¹⁵ The primers detected a methylation hotspot 200 bp upstream from the MGMT start site:MGMT sense, 5'-TTT GTGTTTTGATGTTTGTAGGTTTTTGT-3' and MGMT antisense 5'-AACTCCACACTCTTC CAAAAACAAAACA-3' for unmethylated sequences; MGMT sense 5'-TTTCGACGTTCGTAGGTTTTCGC-3' and MGMT antisense 5'-GCACTCTTCCGAAAACGAAACG-3' for methylated sequences.

MSI Assay
Genomic DNA from pretreatment blasts was extracted and amplified using three MSI multiplex reaction mixtures containing NCI panel markers (BAT-25, BAT-26, D2S123, D17S250, D5S346),³⁰ quasimonomorphic mononucleotide markers (NR-21, NR-22, NR-24, BAT-25, BAT-26),³¹ and an alternative panel of markers (D18S35, TP53-DI, TP53-PENTA, D1S2883, and FGA).³² Tumor cell MSI was compared with peripheral blood or nonmalignant lymphocytes obtained from the same patient. Control, nonmalignant lymphocytes were obtained by flow cytometry from either peripheral blood or bone marrow aspirates, sorting for CD45_bright cells lacking tumor-specific antigens. Each sample was scored according to number of markers positive or negative for MSI. Tumors were categorized into one of three categories; MSI-high (> 40% of markers unstable), MSI-low (less than 40% of markers unstable), or MSI-stable using NCI standards.³³

Statistics
The Wilcoxon rank sum test was used to compare MGMT activity by leukemia subtype (ALL v AML) and patient disease status (newly diagnosed v relapsed). Three banked specimens had total protein concentrations below the level of quantitation (0.1 mg/mL) and these samples were removed from the MGMT analysis. The association of age, sex, race, and WBC count with MGMT levels was assessed univariately using the Wilcoxon rank sum test. Statistical analyses were applied to patient data and banked samples separately.

	RESULTS

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From July 2004 until April 2006, 16 pediatric patients (eight with AML, eight with ALL) were enrolled (Table 1). Ten patients were not fully assessable for toxicity because they had early PD and either received nonprotocol therapy before completing the first course of temozolomide (n = 8) or died as a result of PD during the first course of treatment (n = 2). None of these patients experienced unusual or severe temozolomide-related toxicities. Three patients were fully assessable for toxicity at each dose level.

Response
Two of the 16 patients had an overall response to one cycle of temozolomide treatment. One 5-year-old male with AML (M1 subtype) treated with 200 mg/m² temozolomide had a PR with a decrease in bone marrow blasts from 60% to 10%; he received four courses of temozolomide before developing PD. A 14-year-old female with AML/myelodysplastic syndrome (M2 subtype) treated with 260 mg/m² temozolomide also had a PR, with a decrease in bone marrow blasts from 56% to 6% after one course of temozolomide. Her parents refused additional treatment and she developed PD 6 weeks after cessation of therapy. Four of 16 patients had PD after the completion of course 1 and the remaining 10 of 16 patients had early PD.

Pharmacokinetics
Pharmacokinetic samples were obtained from 10 patients (Table 2). Temozolomide was absorbed rapidly and eliminated from plasma. On the basis of the noncompartmental analysis, the mean time to maximal concentration was 74 ± 50 minutes and the terminal half-life (t_1/2 el) was 109 ± 24 minutes. The mean V_d was 20 ± 5.2 L/m², and the apparent oral systemic Cl was 107 ± 31 mL/min/m². Both AUC and C_max increased in proportion with dose. Compartmental analysis resulted in a mean elimination t_1/2 of 111 ± 37 minutes. Compartmental and noncompartmental t_1/2, Cl, and V_d were similar.

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Summary of O6-methylguanine–DNA methyltransferase (MGMT) repair activity in an independent set of leukemia samples: MGMT activity was determined in 67 pediatric patients with newly diagnosed acute lymphoblastic leukemia (nALL; n = 21), relapsed ALL (rALL; n = 19), newly diagnosed acute myeloid leukemia (nAML; n = 20), relapsed AML (rAML; n = 7), and peripheral-blood mononuclear cells from healthy volunteers (n = 4). (——) Median values for each group. Differences that are statistically different are noted (Wilcoxon rank sum test).

Assessment of MGMT Promoter Methylation
MGMT promoter methylation correlated with MGMT activity in 11 of 12 patients (Table 3; Fig 2). Patients with MGMT promoter methylation had little or no MGMT activity, whereas patients with an unmethylated MGMT promoter had MGMT activity present. Although the two patients with a PR to temozolomide had methylated MGMT promoters and no MGMT expression, patients with no objective response had both unmethylated (n = 10) and methylated (n = 2) MGMT promoters, suggesting that factors other than MGMT activity can result in temozolomide resistance. MGMT promoter methylation status was concordant in plasma and leukemia blasts in the nine patients in whom both were evaluated.

View larger version (9K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. O6-Methylguanine–DNA methyltransferase (MGMT) promoter methylation patterns in patients treated with temozolomide. Genomic DNA was extracted before temozolomide treatment and MGMT promoter methylation was determined by methylation-specific polymerase chain reaction (PCR). PCR product at 110 bp in methylated PCR reactions is nonspecific. (Upper marker) 100 bp; (lower marker) 75 bp. U, unmethylated, M, methylated, Mar, 50-bp marker, NL, normal lymphocytes; Co, SW-48 colon carcinoma; Leu, U937 acute myeloid leukemia cells; PD, progressive disease; PR, partial response.

View larger version (22K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Microsatellite instability (MSI) in pediatric leukemia: (A) microsatellite alterations from a patient demonstrating MSI in the leukemia cells (T), but not the normal lymphocytes (B) for MSI markers BAT-25, BAT-26, D2S123 NR-21, NR-22, and NR-24.31,32 (B) MSI in 65 pediatric leukemia samples categorized by subtype and relapse status. Bars with no numbers represent one patient. nALL, newly diagnosed acute lymphoblastic leukemia; rALL, relapsed ALL; nAML, newly diagnosed acute myeloid leukemia; rAML, relapsed AML; NCI, National Cancer Institute; Quasi, quasimonomorphic mononucleotide markers.

	DISCUSSION

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Most pharmacokinetic parameters were similar to those seen in adults. AUC, t_1/2, V_d, Cl, and C_max values were similar to those reported in both adults and pediatric patients.^34-37 The average T_max occurred slightly later than that reported in adults, but was similar to other pediatric studies.³⁴

Another objective of this trial was to examine the mechanisms of temozolomide resistance. Increased MGMT enzymatic activity has been correlated with temozolomide resistance in both gliomas and leukemias.^1,3,39 Patients in this study with elevated MGMT activity were also resistant to temozolomide. MGMT expression is decreased by MGMT promoter methylation, which is common in many tumor types, and MGMT methylation may predict temozolomide sensitivity.⁴⁰ However, others have found only a moderate association between MGMT promoter methylation and MGMT repair activity.^41-43 In our study, 11 of 12 samples showed concordance between MGMT activity and MGMT promoter methylation. One patient with a methylated MGMT promoter had low MGMT expression. Although likely due to the presence of nonmalignant lymphocytes in this sample, other investigators have noted that MGMT promoter methylation can be associated infrequently with residual MGMT activity.³³

We also noted a concordance between MGMT methylation in tumor cells and plasma. If plasma MGMT promoter methylation accurately reflects tissue MGMT promoter methylation, this assay might have several clinical applications. Since only tumor DNA is abnormally methylated, plasma MGMT methylation could be used to monitor disease response and to detect early relapse in patients whose tumors have methylated MGMT promoters.⁴⁴ Balana et al¹⁵ reported a correlation between plasma MGMT promoter methylation and chemotherapy response in patients with glioblastoma multiforme, suggesting that additional research in this area is warranted.

Some tumors remain resistant to temozolomide despite low MGMT activity due to MRS pathway defects,⁴⁵ which may arise from mutations or gene silencing of MRS proteins.²² MRS defects and the resulting MSI, which are common in leukemia cell lines,^21,23,46,47 are uncommon in primary pediatric leukemias (approximately 10% prevalence)^46,48-50 and in adults with newly diagnosed leukemia.^51,52 However, MSI is more prevalent in relapsed leukemia,^53-55 treatment-related AML,^56-58 and adult T-cell ALL.⁵⁹ In our study, only one patient was MSI-high. Given that several patients with PD had stable MSI (Table 3), it did not seem that MSI was a major determinant of temozolomide resistance. However, MSI stability may be necessary for temozolomide sensitivity, given that leukemia cells with MRS defects are resistant to temozolomide despite low MGMT activity.³

In summary, we determined that in children with relapsed leukemia, temozolomide could be tolerated at doses of 260 mg/m²/d for 5 days. In this heavily pretreated population, however, we did not observe significant clinical activity. Given that patients with relapsed AML in general had lower MGMT activity than patients with relapsed ALL, additional study of temozolomide in this leukemia subtype should be considered. The addition of MGMT inhibitors such as O⁶-benzylguanine to temozolomide might enhance temozolomide efficacy in leukemia.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Terzah M. Horton, Peter C. Adamson, M. Eileen Dolan, Susan M. Blaney

Financial support: Terzah M. Horton, M. Eileen Dolan

Administrative support: Peter C. Adamson

Provision of study materials or patients: Terzah M. Horton, M. Eileen Dolan, Peter C. Adamson, Susan M. Blaney

Collection and assembly of data: Terzah M. Horton, Ashish M. Ingle, M. Eileen Dolan, Shannon M. Delaney, Madhuri Hedge, Susan M. Blaney

Data analysis and interpretation: Terzah M. Horton, Patrick A. Thompson, Stacey L. Berg, Peter C. Adamson, Ashish M. Ingle, M. Eileen Dolan, Shannon M. Delaney, Madhuri Hedge, Heidi L. Weiss, Meng-Fen Wu, Susan M. Blaney

Manuscript writing: Terzah M. Horton, Patrick A. Thompson, Stacey L. Berg, Peter C. Adamson, Ashish M. Ingle, M. Eileen Dolan, Shannon M. Delaney, Susan M. Blaney

Final approval of manuscript: Peter C. Adamson, M. Eileen Dolan, Susan M. Blaney

	ACKNOWLEDGMENTS

 
We thank Anu Gannavarapu, Gaye Jenkins, John Hyatt, and Alexander Aleksic for technical assistance; and Elizabeth O'Connor, Carrianne Hanson, and Shanila Faghfoor of the Cooperative Oncology Group Phase I/Pilot Consortium Coordinating Center for outstanding clinical trial administrative support throughout the development, conduct, and analysis of this study.

	NOTES

 
Supported by National Cancer Institute Grants No. UO1-CA97452 (C.O.G.), U01CA63187 (University of Chicago Cancer Research Center [UCCRC]), K12CA90433 (T.M.H.), The Lady Tata Memorial Fund (T.M.H.), the Scott Carter National Childhood Cancer Foundation Research Fellowship (T.M.H.). Pharmacologic studies were supported by the UCCRC Pharmacology Core Facility (http://pharmacology.bsd.uchicago.edu/) through the UCCRC Cancer Center Support Grant, P30 CA14599.

Presented in abstract form at the American Society of Hematology Meeting, San Diego, CA, December 10-15, 2005.

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

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Submitted April 5, 2007; accepted August 13, 2007.

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