Phase I pharmacokinetic and pharmacodynamic study of temozolomide in pediatric patients with refractory or recurrent leukemia: a Children's Oncology Group Study

Abstract

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).

Figure 1:. Summary of MGMT enzyme activity in an independent set of leukemia samples:

MGMT activity was determined in 67 pediatric patients with newly-diagnosed ALL (nALL)(n=21), relapsed ALL (rALL) (n=19), newly-diagnosed AML (nAML) (n=20), relapsed AML (rAML)(n=7), and PBMC from healthy volunteers (n=4). Median values for each group noted as solid line. Differences that are statistically different are noted (Wilcoxon rank-sum test).

Figure 2:. MGMT promoter methylation patterns in patients treated with temozolomide.

Genomic DNA was extracted prior to temozolomide treatment and MGMT promoter methylation determined by methylation-specific PCR. U= unmethylated, M=methylated, Mar= 50bp marker, NL= normal lymphocytes, Co- SW-48 colon carcinoma, Leu = U937 AML cells. PD = progressive disease, PR = partial response. PCR product at 110 bp in methylated PCR reaction is non-specific.

Figure 3:. Microsatellite instability in pediatric leukemia:

(A) Representative fluorescent-based microsatellite alterations from a patient demonstrating microsatellite instability in the leukemia cells (T), but not the normal lymphocytes (B) for the NCI MSI markers BAT-25, BAT-26, and D2S123 and the quasimonomorphic MSI markers NR-21, NR-22 and NR-24. (B) Microsatellite instability was assessed in 65 pediatric leukemia samples categorized by subtype and relapse status. Microsatellite instability was determined at 13 alleles. Samples were classified as stable, having instability at a single site (MSI-low), or having instability at multiple sites (MSI-high), consistent with a mutator phenotype (RER+).