A molecular biology and phase II study of imetelstat (GRN163L) in children with recurrent or refractory central nervous system malignancies: a pediatric brain tumor consortium study

Abstract

Telomerase activation is critical in many cancers including central nervous system (CNS) tumors. Imetelstat is an oligonucleotide that binds to the template region of the RNA component of telomerase, inhibiting its enzymatic activity. We conducted an investigator-sponsored molecular biology (MB) and phase II study to estimate inhibition of tumor telomerase activity and sustained responses by imetelstat in children with recurrent CNS malignancies. In the MB study, patients with recurrent medulloblastoma, high-grade glioma (HGG) or ependymoma undergoing resection received one dose of imetelstat as a 2-h intravenous infusion at 285 mg/m(2), 12-24 h before surgery. Telomerase activity was evaluated in fresh tumor from surgery. Post-surgery and in the phase II study, patients received imetelstat IV (days 1 and 8 q21-days) at 285 mg/m(2). Imetelstat pharmacokinetic and pharmacodynamic studies were performed. Of two evaluable patients on the MB trial, intratumoral telomerase activity was inhibited by 95 % compared to baseline archival tissue in one patient and was inevaluable in one patient. Forty-two patients (40 evaluable for toxicity) were enrolled: 9 medulloblastomas, 18 HGG, 4 ependymomas, 9 diffuse intrinsic pontine gliomas. Most common grade 3/4 toxicities included thrombocytopenia (32.5 %), lymphopenia (17.5 %), neutropenia (12.5 %), ALT (7.5 %) and AST (5 %) elevation. Two patients died of intratumoral hemorrhage secondary to thrombocytopenia leading to premature study closure. No objective responses were observed. Telomerase inhibition was observed in peripheral blood mononuclear cells (PBMCs) for at least 8 days. Imetelstat demonstrated intratumoral and PBMC target inhibition; the regimen proved too toxic in children with recurrent CNS tumors.

Keywords: Imetelstat; Pediatric brain tumors; Phase 2 trial; Telomerase; Telomerase inhibition.

Figure 1

(A) Patient received imetelstat on days 1 and 8 in each 21-day cycle at 285mg/m². Total protein extracts were assayed for telomerase activity during course 1, day 1 pre-IMT (C1D1-pre); course 1, day 1 post-IMT (C1D1-post); course 1, day 2 (C1D2); course 1, day 8 (C1D8); course 2, day 1 pre-IMT (C2D1) and course 3, day 1 pre-IMT (C3D1). Telomerase activity was evaluated by TRAP assay using 800 ng of total protein extracted from PBMCs. Lanes labeled (−ve) and (+ve) depict the negative (protein extraction buffer) and the positive controls (30 ng of total protein extract from HeLa cells) of the assay, respectively. (B) Quantification of telomerase activity in (A). The telomerase products (6-bp ladder) and the 36-bp internal control (PCR internal control) bands were quantified using a Storm phosphoimager. Relative telomerase activity was calculated as the intensity ratio of the TRAP ladder (telomerase products) to that of the PCR internal control. The percentage of inhibition was calculated by dividing the intensity ratio of sample post-IMT with the intensity ratio of the sample pre-IMT at different timepoints: course 1, day 1 pre-IMT (C1D1-pre); course 1, day 1 post-IMT (C1D1-post); course 1, day 2 (C1D2); course 1, day 8 (C1D8); course 2, day 1 pre-IMT (C2D1) and course 3, day 1 pre-IMT (C3D1). Note sustained inhibition of telomerase activity until C1D8 and recovery of telomerase activity by C2D1.

Figure 2

In tumor inhibition of telomerase activity post-imetelstat (IMT) treatment in one patient with HGG enrolled on the MB study. (A) Patient received the first dose of imetelstat 26.5 hours before surgery. Total protein extracts were assayed for telomerase activity. Telomerase activity was evaluated by TRAP assay using 400 ng of total protein extracted from fresh flash-frozen tumor tissue samples collected from archival tissue (lane 1, pre-IMT) and second surgery (lane 2, post-IMT). Lanes 3 and 4 are the negative (protein extraction buffer) and the positive controls (30 ng of total protein extract from HeLa cells) of the assay, respectively. PCR internal control and telomerase products are indicated with a black arrow. (B) Quantification of telomerase activity in (A). The telomerase products (6-bp ladder) and the 36-bp internal control (PCR internal control) bands were quantified using a Storm phosphoimager. Relative telomerase activity was calculated as the intensity ratio of the TRAP ladder (telomerase products) to that of the PCR internal control. The percentage of inhibition was calculated by dividing the intensity ratio of sample post-IMT with the intensity ratio of the sample pre-IMT. The assay was repeated 3 times. The calculated telomerase activity inhibition is ~95% relative to telomerase activity in the tissue collected at the time of the first surgery (lane 1). The values shown are the mean +/− SD of 3 independent experiments.