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Journal of Clinical Oncology, Vol 25, No 31 (November 1), 2007: pp. 4922-4928
© 2007 American Society of Clinical Oncology.
DOI: 10.1200/JCO.2007.12.0667
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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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 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 REFERENCES
 
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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 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 REFERENCES
 
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 leukemia11,12; 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 tumors9,15 and diffuse large B-cell lymphomas.16 However some patients with leukemia have evidence of MGMT promoter hypermethylation17,18 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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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 REFERENCES
 
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 (Tmax), peak plasma concentration (Cmax), area under the concentration-time curve (AUC), and apparent volume of distribution at steady state (Vd-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 CD45bright 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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 RESULTS
DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
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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.


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  		Table 1. Characteristics for Eligible Patients (n = 16)

 
Toxicity
Temozolomide was well tolerated in children with recurrent or refractory leukemia. There were no DLTs at the two dose levels evaluated. Non-DLTs related to temozolomide included one occurrence each of grade 3 nausea, vomiting, fever with neutropenia, pneumonia, and elevated serum aminotransferase levels.

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 (t1/2 el) was 109 ± 24 minutes. The mean Vd was 20 ± 5.2 L/m2, and the apparent oral systemic Cl was 107 ± 31 mL/min/m2. Both AUC and Cmax increased in proportion with dose. Compartmental analysis resulted in a mean elimination t1/2 of 111 ± 37 minutes. Compartmental and noncompartmental t1/2, Cl, and Vd were similar.


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  		Table 2. Summary of Day 1 Pharmacokinetic Parameters From 10 Pediatric Patients Treated With Temozolomide

 
Assessment of Pretreatment MGMT Repair Activity
We assessed MGMT repair activity in 12 patients (Table 3). Patients had a wide range of MGMT activity (< 100 to > 2,500 fmol/mg protein) compared with normal volunteers, who had an average MGMT activity of 771 ± 170 fmol/mg protein (Fig 1). MGMT activity seemed to correlate with leukemia subtype; three of seven patients with AML had MGMT activity below the level of detection (< 5%), whereas no ALL patients had undetectable MGMT activity. Although four of five patients with ALL had elevated MGMT protein activity (> 1,100 fmol/mg protein), no patients with AML had elevated MGMT activity (Table 3). There was no correlation between patient MGMT activity and age, sex, or race. The two patients with a PR to temozolomide had undetectable MGMT activity.


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  		Table 3. Mechanisms of Temozolomide Resistance: MGMT Activity and MSI in Peripheral Blood or Bone Marrow Aspirate Samples (n = 14)

 

Figure 1
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  		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).

 
We explored MGMT activity further in an independent set of 67 banked ALL and AML samples obtained from the Texas Children's Hospital tissue tumor bank using an IRB-approved protocol. Newly diagnosed patients (ALL, n = 21; AML, n = 21) were chemotherapy naive and relapsed patients (ALL, n = 18; AML, n = 7) had samples obtained before reinduction chemotherapy. As shown in Figure 1, MGMT activity correlated with leukemia subtype. ALL lymphoblasts had significantly higher MGMT activity than did AML myeloblasts (P < .0001). In general, specimens from patients with relapsed ALL had higher MGMT activity than specimens from patients with either newly diagnosed ALL (P = .02) or relapsed AML (P < .01). MGMT activity did not correlate with sex but it seemed to correlate with race (whites, n = 56: 1,316 ± 1,227 fmol/mg protein v nonwhites, n = 10: 623 ± 761 fmol/mg protein; P = .05). Differences in MGMT activity between leukemia subtypes, however, remained significant after controlling for this potential confounder.

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.


Figure 2
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  		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.

 
MSI
Thirteen of the 14 patients tested were MSI-stable; only one patient was MSI-high (Table 3). This patient had MSI at multiple MSI loci (Fig 3A). MSI was also examined in 65 banked leukemia samples. As shown in Figure 3B, samples from patients with newly diagnosed ALL or AML were MSI-stable or MSI-low. In contrast, samples from nine of 18 patients with relapsed ALL were either MSI-low (two of 18) or MSI-high (seven of 18). Several patients with relapsed AML (three of seven) were also MSI-low. This suggests that MSI is common in pediatric patients with relapsed leukemia.


Figure 3
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  		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
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 REFERENCES
 
In this study we examined the toxicities, response, and pharmacokinetics of temozolomide administered as a single agent to pediatric patients with relapsed or refractory leukemia. Temozolomide drug disposition in children with leukemia was similar to that observed in pediatric patients with solid tumors34 as well as in adults.25,35-38 Because of the large number of patients with PD during the first treatment course, the MTD of temozolomide in pediatric patients with relapsed leukemia was not determined. However, three assessable patients tolerated 260 mg/m2 without evidence of DLTs. Of the 16 patients enrolled, two had objective responses.

Most pharmacokinetic parameters were similar to those seen in adults. AUC, t1/2, Vd, Cl, and Cmax values were similar to those reported in both adults and pediatric patients.34-37 The average Tmax 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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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
AUTHOR CONTRIBUTIONS
 REFERENCES
 
The author(s) indicated no potential conflicts of interest.


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


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 REFERENCES
 
1. Friedman HS, Kerby T, Calvert H: Temozolomide and treatment of malignant glioma. Clin Cancer Res 6 : 2585 -2597, 2000[Abstract/Free Full Text]

2. Stupp R, Mason WP, van den Bent MJ, et al: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352 : 987 -996, 2005[Abstract/Free Full Text]

3. Tentori L, Graziani G, Gilberti S, et al: Triazene compounds induce apoptosis in O6-alkylguanine-DNA alkyltransferase deficient leukemia cell lines. Leukemia 9 : 1888 -1895, 1995[Medline]

4. Messinger Y, Reaman GH, Ek O, et al: Evaluation of temozolomide in a SCID mouse model of human B-cell precursor leukemia. Leuk Lymphoma 33 : 289 -293, 1999[Medline]

5. Seiter K, Liu D, Loughran T, et al: Phase I study of temozolomide in relapsed/refractory acute leukemia. J Clin Oncol 20 : 3249 -3253, 2002[Abstract/Free Full Text]

6. Baer JC, Freeman AA, Newlands ES, et al: Depletion of O6-alkylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumour cells. Br J Cancer 67 : 1299 -1302, 1993[Medline]

7. Friedman HS, McLendon RE, Kerby T, et al: DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma. J Clin Oncol 16 : 3851 -3857, 1998[Abstract/Free Full Text]

8. Gerson SL: Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 20 : 2388 -2399, 2002[Abstract/Free Full Text]

9. Esteller M, Garcia-Foncillas J, Andion E, et al: Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343 : 1350 -1354, 2000[Abstract/Free Full Text]

10. Pollack IF, Hamilton RL, Sobol RW, et al: O6-methylguanine-DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: Results from the CCG-945 Cohort. J Clin Oncol 24 : 3431 -3437, 2006[Abstract/Free Full Text]

11. Taverna P, Catapano CV, Citti L, et al: Influence of O6-methylguanine on DNA damage and cytotoxicity of temozolomide in L1210 mouse leukemia sensitive and resistant to chloroethylnitrosoureas. Anticancer Drugs 3 : 401 -405, 1992[Medline]

12. Tentori L, Orlando L, Lacal PM, et al: Inhibition of O6-alkylguanine DNA-alkyltransferase or poly(ADP-ribose) polymerase increases susceptibility of leukemic cells to apoptosis induced by temozolomide. Mol Pharmacol 52 : 249 -258, 1997[Abstract/Free Full Text]

13. Esteller M, Hamilton SR, Burger PC, et al: Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59 : 793 -797, 1999[Abstract/Free Full Text]

14. Costello JF, Futscher BW, Tano K, et al: Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells. J Biol Chem 269 : 17228 -17237, 1994[Abstract/Free Full Text]

15. Balaña C, Ramirez JL, Taron M, et al: O(6)-methyl-guanine-DNA methyltransferase methylation in serum and tumor DNA predicts response to 1,3-bis(2-Chloroethyl)-1-nitrosourea but not to temozolomide plus cisplatin in glioblastoma multiforme. Clin Cancer Res 9 : 1461 -1468, 2003[Abstract/Free Full Text]

16. Esteller M, Gaidano G, Goodman SN, et al: Hypermethylation of the DNA repair gene O(6)-methylguanine DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma. J Natl Cancer Inst 94 : 26 -32, 2002[Abstract/Free Full Text]

17. Rossi D, Capello D, Gloghini A, et al: Aberrant promoter methylation of multiple genes throughout the clinico-pathologic spectrum of B-cell neoplasia. Haematologica 89 : 154 -164, 2004[Abstract/Free Full Text]

18. Matsushita C, Yang Y, Takeuchi S, et al: Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia. Oncol Rep 12 : 97 -99, 2004[Medline]

19. D'Atri S, Piccioni D, Castellano A, et al: Chemosensitivity to triazene compounds and O6-alkylguanine-DNA alkyltransferase levels: Studies with blasts of leukaemic patients. Ann Oncol 6 : 389 -393, 1995[Abstract/Free Full Text]

20. Liu L, Markowitz S, Gerson SL: Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea. Cancer Res 56 : 5375 -5379, 1996[Abstract/Free Full Text]

21. Hangaishi A, Ogawa S, Mitani K, et al: Mutations and loss of expression of a mismatch repair gene, hMLH1, in leukemia and lymphoma cell lines. Blood 89 : 1740 -1747, 1997

22. Taverna P, Liu L, Hanson AJ, et al: Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen. Cancer Chemother Pharmacol 46 : 507 -516, 2000[CrossRef][Medline]

23. Gu L, Cline-Brown B, Zhang F, et al: Mismatch repair deficiency in hematological malignancies with microsatellite instability. Oncogene 21 : 5758 -5764, 2002[CrossRef][Medline]

24. Shen F, Decosterd LA, Gander M, et al: Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction. J Chromatogr B Biomed Appl 667 : 291 -300, 1995[CrossRef][Medline]

25. Rudek MA, Donehower RC, Statkevich P, et al: Temozolomide in patients with advanced cancer: Phase I and pharmacokinetic study. Pharmacotherapy 24 : 16 -25, 2004[CrossRef][Medline]

26. DiArgenio DZ, Schumitsky A: ADAPT II Users Guide: Pharmacokinetic/Pharmacodynamic Systems Analysis Software. Los Angeles, CA, Biomedical Systems Resource (version 4), 1997

27. Yamaoka K, Nakagawa T, Uno T: Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm 6 : 165 -175, 1978[CrossRef][Medline]

28. Horton TM, Pati D, Plon SE, et al: A phase 1 study of the proteasome inhibitor bortezomib in pediatric patients with refractory leukemia: A Children's Oncology Group Study. Clin Cancer Res 13 : 1615 -1622, 2007

29. Dolan ME, Roy SK, Fasanmade AA, et al: O6-benzylguanine in humans: Metabolic, pharmacokinetic, and pharmacodynamic findings. J Clin Oncol 16 : 1803 -1810, 1998[Abstract]

30. Boland CR, Thibodeau SN, Hamilton SR, et al: A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58 : 5248 -5257, 1998[Abstract/Free Full Text]

31. Suraweera N, Duval A, Reperant M, et al: Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123 : 1804 -1811, 2002[CrossRef][Medline]

32. Frazier ML, Sinicrope FA, Amos CI, et al: Loci for efficient detection of microsatellite instability in hereditary non-polyposis colorectal cancer. Oncol Rep 6 : 497 -505, 1999[Medline]

33. Goel A, Arnold CN, Niedzwiecki D, et al: Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res 63 : 1608 -1614, 2003[Abstract/Free Full Text]

34. Estlin EJ, Lashford L, Ablett S, et al: Phase I study of temozolomide in paediatric patients with advanced cancer: United Kingdom Children's Cancer Study Group. Br J Cancer 78 : 652 -661, 1998[Medline]

35. Dhodapkar M, Rubin J, Reid JM, et al: Phase I trial of temozolomide (NSC 362856) in patients with advanced cancer. Clin Cancer Res 3 : 1093 -1100, 1997[Abstract]

36. Hammond LA, Eckardt JR, Baker SD, et al: Phase I and pharmacokinetic study of temozolomide on a daily-for-5-days schedule in patients with advanced solid malignancies. J Clin Oncol 17 : 2604 -2613, 1999[Abstract/Free Full Text]

37. Brada M, Judson I, Beale P, et al: Phase I dose-escalation and pharmacokinetic study of temozolomide (SCH 52365) for refractory or relapsing malignancies. Br J Cancer 81 : 1022 -1030, 1999[CrossRef][Medline]

38. Reid JM, Stevens DC, Rubin J, et al: Pharmacokinetics of 3-methyl-(triazen-1-yl)imidazole-4-carboximide following administration of temozolomide to patients with advanced cancer. Clin Cancer Res 3 : 2393 -2398, 1997[Abstract/Free Full Text]

39. Tisdale MJ: Antitumor imidazotetrazines: XV. Role of guanine O6 alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem Pharmacol 36 : 457 -462, 1987[CrossRef][Medline]

40. Gerson SL: MGMT: Its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4 : 296 -307, 2004[CrossRef][Medline]

41. Yeager ND, Dolan ME, Gastier JM, et al: O6-methylguanine-DNA methyltransferase activity and promoter methylation status in pediatric rhabdomyosarcoma. J Pediatr Hematol Oncol 25 : 941 -947, 2003[CrossRef][Medline]

42. Nakayama K, Inokuchi K, Dan K: Hypermethylation of the putative tumor-suppressor genes DCC, p51/63 and O6-methylguanine-DNA methyltransferase (MGMT) and loss of their expressions in cell lines of hematological malignancies. J Nippon Med Sch 72 : 270 -277, 2005[CrossRef][Medline]

43. Pieper RO, Patel S, Ting SA, et al: Methylation of CpG island transcription factor binding sites is unnecessary for aberrant silencing of the human MGMT gene. J Biol Chem 271 : 13916 -13924, 1996[Abstract/Free Full Text]

44. Soejima H, Zhao W, Mukai T: Epigenetic silencing of the MGMT gene in cancer. Biochem Cell Biol 83 : 429 -437, 2005[CrossRef][Medline]

45. Levati L, Marra G, Lettieri T, et al: Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents. Genes Chromosomes Cancer 23 : 159 -166, 1998[CrossRef][Medline]

46. Ribeiro EM, Rodriguez JM, Coser VM, et al: Microsatellite instability and cytogenetic survey in myeloid leukemias. Braz J Med Biol Res 35 : 153 -159, 2002[Medline]

47. Kodera T, Kohno T, Takakura S, et al: Microsatellite instability in lymphoid leukemia and lymphoma cell lines but not in myeloid leukemia cell lines. Genes Chromosomes Cancer 26 : 267 -269, 1999[CrossRef][Medline]

48. Inokuchi K, Ikejima M, Watanabe A, et al: Loss of expression of the human MSH3 gene in hematological malignancies. Biochem Biophys Res Commun 214 : 171 -179, 1995[CrossRef][Medline]

49. Takeuchi S, Seriu T, Tasaka T, et al: Microsatellite instability and other molecular abnormalities in childhood acute lymphoblastic leukaemia. Br J Haematol 98 : 134 -139, 1997[CrossRef][Medline]

50. Reato G, Basso G, Putti MC, et al: Microsatellite analysis in childhood acute lymphoblastic leukemia. Haematologica 83 : 403 -407, 1998[Abstract/Free Full Text]

51. Krsková-Honzátková L, Cermak J, Sajdova J, et al: Microsatellite instability in hematological malignancies. Leuk Lymphoma 43 : 1979 -1986, 2002[CrossRef][Medline]

52. Ohyashiki JH, Ohyashiki K, Aizawa S, et al: Replication errors in hematological neoplasias: Genomic instability in progression of disease is different among different types of leukemia. Clin Cancer Res 2 : 1583 -1589, 1996[Abstract]

53. Tasaka T, Lee S, Spira S, et al: Microsatellite instability during the progression of acute myelocytic leukaemia. Br J Haematol 98 : 219 -221, 1997[CrossRef][Medline]

54. Uehara E, Takeuchi S, Tasaka T, et al: Aberrant methylation in promoter-associated CpG islands of multiple genes in therapy-related leukemia. Int J Oncol 23 : 693 -696, 2003[Medline]

55. Das-Gupta EP, Seedhouse CH, Russell NH: Microsatellite instability occurs in defined subsets of patients with acute myeloblastic leukaemia. Br J Haematol 114 : 307 -312, 2001[CrossRef][Medline]

56. Casorelli I, Offman J, Mele L, et al: Drug treatment in the development of mismatch repair defective acute leukemia and myelodysplastic syndrome. DNA Repair (Amst) 2 : 547 -559, 2003[CrossRef][Medline]

57. Sheikhha MH, Tobal K, Liu Yin JA: High level of microsatellite instability but not hypermethylation of mismatch repair genes in therapy-related and secondary acute myeloid leukaemia and myelodysplastic syndrome. Br J Haematol 117:359-365, 2002

58. Olipitz W, Hopfinger G, Aguiar RC, et al: Defective DNA-mismatch repair: A potential mediator of leukemogenic susceptibility in therapy-related myelodysplasia and leukemia. Genes Chromosomes Cancer 34 : 243 -248, 2002[CrossRef][Medline]

59. Hatta Y, Yamada Y, Tomonaga M, et al: Microsatellite instability in adult T-cell leukaemia. Br J Haematol 101 : 341 -344, 1998[CrossRef][Medline]

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




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