A Phase 1 Trial of Imetelstat in Children with Refractory or Recurrent Solid Tumors: A Children’s Oncology Group Phase 1 Consortium Study (ADVL1112)

Abstract

Purpose

Imetelstat is a covalently-lipidated 13-mer thiophosphoramidate oligonucleotide that acts as a potent specific inhibitor of telomerase. It binds with high affinity to the template region of the RNA component of human telomerase (hTERC ) and is a competitive inhibitor of telomerase enzymatic activity. The purpose of this study was to determine the recommended phase 2 dose of imetelstat in children with recurrent or refractory solid tumors.

Experimental Design

Imetelstat was administered intravenously over two hours on days 1 and 8, every 21 days. Dose levels of 225, 285, and 360 mg/m² were evaluated, using the rolling-six design. Imetelstat pharmacokinetic and correlative biology studies were also performed during the first cycle.

Results

Twenty subjects were enrolled (median age 14 yrs; range 3–21). Seventeen were evaluable for toxicity. The most common toxicities were neutropenia, thrombocytopenia, and lymphopenia, with dose-limiting myelosuppression in two of six patients at 360 mg/m². Pharmacokinetics were dose dependent with a lower clearance at the highest dose level. Telomerase inhibition was observed in peripheral blood mononuclear cells at 285 and 360 mg/m². Two confirmed partial responses osteosarcoma (n=1) and Ewing sarcoma (n=1) were observed.

Conclusions

The recommended phase 2 dose of imetelstat given on days 1 and 8 of 21-day cycle is 285 mg/m².

INTRODUCTION

Telomeres, specialized structures found at the end of chromosomes, are involved in the replication and stability of the chromosome. Telomeres consist of tandem repeats of the DNA sequence TTAGGG and associated proteins. During the process of cell division, most human cells undergo telomere shortening because they lose some of these tandem repeats, approximately 50–200 base pairs (bp) per cell division.(1, 2) When telomeres become critically short, cells either becomes senescent or undergo apoptosis.

The enzyme telomerase plays an important role in the formation, maintenance, and renovation of telomeres. Telomerase, or telomere terminal transferase, is a ribonucleoprotein that catalyzes the de novo synthesis and elongation of telomeric repeats at chromosomal ends by using an RNA segment within the RNA subunit as a template.(3–5) Telomerase consists of at least two essential components, the RNA template (hTERC) and the catalytic subunit (hTERT).

Cancer development is accompanied by the preservation of telomere length, which in most cases results from the reactivation of telomerase.(6, 7) This reactivation is believed to be critical for tumor progression because it enables cancer cells to maintain their telomere length and avoid apoptosis.(8) Approximately 90% of biopsies from a range of human cancers have been found to express telomerase activity,(7, 9, 10) including a wide variety of pediatric tumors such as hepatoblastoma, Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma.(11) Furthermore correlations between tumor stage and telomerase activity have been observed, with early stage tumors having less telomerase activity than late stage tumors.(10, 12–14) Based on the high level of telomerase expression common to most cancers and their relatively shorter telomeres compared to their normal tissue counterparts, along with a low expectation of major toxicities occurring in normal tissues, telomerase is a rational target for the treatment of cancer with potentially broad applicability.

Imetelstat has demonstrated broad activity in vitro and in vivo against a variety of tumor types. Inhibition of xenograft tumor growth and metastases in rodents at plasma exposures that overlap with plasma exposures attained in patients participating in imetelstat clinical trials have been demonstrated in breast cancer, myeloma, small cell lung cancer, and non-small cell lung cancer models.(15–17) Evidence also suggests that telomerase inhibition is a potential candidate for targeted therapy in pediatric brain tumors. In malignant gliomas, telomerase is positive in 10 to 100% of anaplastic astrocytomas and in 26 to 100% of GBM.(18) In 76% of primary medulloblastomas and other primitive neuroectodermal brain tumors have upregulated hTERT mRNA expression compared to normal human cerebellum.(19) Imetelstat has also been observed to cross the blood-brain barrier in an orthotopic glioblastoma multiforme (GBM) mouse model. Tumor cells isolated from the orthotopic tumors following systemic administration of imetelstat showed approximately 70% inhibition of telomerase activity.(20)

Imetelstat was administered as a single agent in three phase 1 studies in adults (advanced solid tumors, multiple myeloma, chronic lymphoproliferative diseases) with the most activity observed in essential thrombocythemia and multiple myeloma.(21) The recommended phase 2 dose and schedule for further testing of imetelstat as a single agent in adults is 9.4 mg/kg on days 1 and 8 of a 21-day cycle.(22) In the solid tumor patient study hematologic toxicity was dose limiting; cytopenias were unacceptable at 11.7 mg/kg. However, at 9.4 mg/kg, 12 patients were treated without first cycle dose-limiting toxicity.(22)

We report the results of a phase I trial of imetelstat in children with recurrent or refractory solid tumors. The primary objectives of this trial were to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose, define and describe the toxicities, and characterize the pharmacokinetics of imetelstat. We also assessed the biologic activity by analyzing peripheral blood mononuclear cell extracts for telomerase activity, hTERT, hTERC expression and telomere length prior to and following imetelstat therapy.

METHODS

Patient Eligibility

Patients older than 12 months and younger than 22 years with measurable or evaluable recurrent or refractory solid tumors were eligible for enrollment. Histologic verification of malignancy from either the time of original diagnosis or relapse was required. Other eligibility criteria included: Lansky or Karnofsky score ≥ 60; recovery from the acute toxic effects of prior therapy; ≥ 6 months since total body irradiation, craniospinal or hemi-pelvic radiation; and ≥ 3 months since a stem cell transplant; adequate bone marrow function for patients with solid tumors (peripheral absolute neutrophil count ≥ 1,000/μL, platelets ≥ 100,000/μL (transfusion independent), hemoglobin ≥ 8.0 g/dL; adequate renal function (age-adjusted normal serum creatinine or a glomerular filtration rate ≥ 70 mL/min/1.73 m²); adequate liver function [total bilirubin ≤ 1.5× institutional upper limit of normal for age, ALT ≤ 110 U/L and albumin ≥ 2 g/dL]; adequate coagulation (PTT ≤1.2 x upper limit of normal). Patients with solid tumors with known bone marrow metastatic disease were eligible but were not evaluable for hematologic toxicity. Patients were excluded if they were pregnant or lactating or if they had uncontrolled infections.

This trial was approved by the institutional review board of each participating institution. Written informed consent and assent, as appropriate, were obtained in accordance with federal and institutional guidelines.

Drug Administration

Imetelstat was supplied by Geron Corporation (Menlo Park, CA) as a white to pale yellow sterile lyophilized powder. Drug was reconstituted with 0.9% sodium chloride for injection to yield a reconstituted drug concentration of 33.33 mg/mL. An appropriate amount of reconstituted drug was then added to a sufficient volume of 0.9% sodium chloride for injection to achieve a final imetelstat concentration of 1 mg/mL for the 225 mg/m² and 285 mg/m² dose levels and to a final concentration of 1.3 mg/ml for the 360 mg/m² dose level. Drug was administered over two hours.

The starting dose of imetelstat was 225 mg/m² (equivalent to approximately 80% of the adult recommended dose of 9.4 mg/kg) with dose escalations in ~30% increments to 285 mg/m² and 360 mg/m². De-escalation of imetelstat to 165 mg/m² was planned if dose-limiting toxicity was observed at the starting dose level.

Study Design

Briefly, up to six patients were enrolled concurrently at the starting dose. Enrollment to subsequent dose levels was determined by the number of enrolled patients, the number with dose limiting toxicity (DLT), and the number at risk for DLT using the rolling-six design (23).

Toxicity was graded according to the Common Terminology Criteria for Adverse Events version 4.0 (http://ctep.cancer.gov). Hematologic DLT was defined as grade 4 neutropenia for > 7 days; grade 4 thrombocytopenia on two separate days, or requiring a platelet transfusion on two separate days within a seven day period; or myelosuppression that caused a delay of > 14 days between treatment cycles. Nonhematologic DLT was defined as grade 3 or 4 nonhematologic toxicity attributable to the investigational drug with the exclusion of grade 3 nausea and vomiting of fewer than three days duration; grade 3 transaminase elevation that returned to ≤ Grade 1 or baseline prior to the time for the next treatment cycle; grade 3 fever or infection; or grade 3 electrolyte abnormalities responsive to oral supplementation. Nonhematologic DLTs included any nonhematologic toxicity that caused a delay of ≥ 14 days between treatment cycles.

Tumor response was reported using the Response Evaluation Criteria in Solid Tumors (RECIST).(24)

Study Evaluations

Patient history, physical examination, and laboratory studies were obtained before treatment and then weekly throughout the first cycle of therapy and before subsequent courses thereafter. CBCs were obtained at least twice weekly during the first cycle and weekly thereafter. Disease evaluations were obtained at baseline, at the end of cycle 1, and after every other cycle x two and then after every third cycle.

Pharmacokinetic Studies

Sample Collection

Blood samples (3 mL for patients > 10 kg, 2 mL for patients ≤ 10 kg) for imetelstat pharmacokinetic studies were placed in EDTA tubes. Samples were collected at the end of infusion and at 0.5, 1, 2, 4, 6–8, and 24 (± 2) hours after the infusion following the first imetelstat dose. A 48 hour sample was also obtained in consenting patients. Serum was separated by centrifugation at 1200–1500 RCF for a minimum of 10 minutes, transferred into cryogenic tubes a polypropylene tube, and stored at −70°C until analysis.

Sample Analysis

Plasma imetelstat concentrations were measured using a validated hybridization-ELISA assay. The lower limit of quantitation for imetelstat in human plasma was 367 ng/mL, and the assay dynamic range was between 367 ng/mL and 2448 ng/mL. The inter-day accuracy was 97.9–104.3% with a coefficient of variation of 5.2–9.8%. Pharmacokinetic parameters for imetelstat were calculated using noncompartmental analysis with WinNonlin Enterprise, version 5.2 (Pharsight Corporation, Mountain View, CA).

Biologic Assays

Analysis of Telomerase Activity in Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were isolated from patients’ whole blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Uppsala, Sweden) according to the manufacturer’s instructions. Briefly, 1X room temperature phosphate buffered saline; PBS without Ca²⁺, Mg²⁺ (Hyclone laboratories Inc., Logan, Utah) was added to the whole blood to a total volume of 15 ml. This mixture was slowly added to a 50 ml conical tube containing 13 ml of Ficoll-Paque PLUS and centrifuged at 400 g for 30 min at room temperature. After centrifugation, the PBMC containing layer was collected and washed 3 times with 3 volumes of room temperature 1X Hank’s balanced salt solution, HBSS (Mediatech, Manassas, Virginia) and centrifuged a second time at 365 g for 10 min at 20°C. The cells were resuspended in 3 ml cold PBS and the cell count and viability were determined. Cells were then pelleted at 3200xg, at 4°C for 1 min, and cell pellets were flash-frozen in dry ice and immediately stored at −80°C until assays were performed. PBMC were collected prior to treatment; 6–8 hours, and 24 (± 2 hr) hours after the first imetelstat dose; prior to the imetelstat dose on Day 8; and prior to the imetelstat dose on Day 1 of Cycles 2 and 3. PBMC extracts were analyzed for telomerase activity prior to and following therapy with imetelstat. Telomerase enzyme activity was assessed using the TRAPeze Telomerase detection and TRAPeze XL telomerase detection kits (Millipore, Billerica, MA). Cell extracts were prepared according to protocols provided by the manufacturer and 400 ng of total protein was assayed for telomerase activity.

RESULTS

Twenty patients were enrolled on study between June 2011 and April 2012. Three patients were not fully evaluable for toxicity because they did not complete Cycle 1. One of the three patients was removed for progressive disease. One withdrew before starting treatment and one withdrew (patient preference) before Day 8 drug administration. Patients received a median of one cycle of therapy (range, 1 to 8). Patient characteristics are summarized in Table 1.

Table 1.

Characteristics for Eligible Patients (n=20)

Characteristic	Number (%)
Age (years)
Median	14.9
Range	3.4–21.9
Sex
Male	13 (65)
Female	7 (35)
Race
White	14 (70)
Asian	1 (5)
American Indian or Alaska Native	0 (0)
Black or African American	1 (5)
Unknown	4 (20)
Ethnicity
Non-Hispanic	15 (75)
Hispanic	4 (20)
Unknown	1 (5)
Diagnoses
Ewing sarcoma	6 (30)
Neuroblastoma	6 (30)
Hepatocellular carcinoma, fibrolamellar	2 (10)
Other1	6 (30)
Prior Therapy
Chemotherapy Regimens
Median	3
Range	0–8
Number of Patients with Prior Radiation Therapy	11

¹Includes one patient each with: Wilm tumor, adrenal cortical carcinoma, embryonal rhabdomyosarcoma, osteosarcoma, alveolar soft part sarcoma, and Hodgkin lymphoma.

Toxicity

Table 2 summarizes the observed DLTs. At the third dose level (360 mg/m²), one patient experienced thrombocytopenia leading to a delay in treatment of more than 14 days and a second patient experienced both neutropenia and thrombocytopenia leading to a delay in therapy of more than 14 days.

Table 2.

Summary of Dose Limiting Toxicities

Dose Level	Number of Patients Entered	Number of Patients Evaluable	Number of pts with DLT	Type of DLT(n)
225 mg/m2	7	5	0
285 mg/m2	7	6	1	Platelet count decreased (1)
360 mg/m2	6	6	2	Neutrophil count decreased (1), Platelet count decreased (2)

Table 3 summarizes adverse events ≥ grade 3 at least possibly attributable to imetelstat in the 17 evaluable patients.

Table 3.

Toxicities (≥ Grade 3) Observed in Evaluable Patients & Attributed at Least Possibly Related to Drug

Toxicity Type	Maximum grade per patient
	(Cycle 1, total 17 courses)		(Cycles 2–8, total 17 courses)
	Grade 3	Grade 4	Grade 3	Grade 4
Anemia	1	0	2	0
Lymphopenia	1	0	1	0
Neutropenia	0	0	0	1
Thrombocytopenia	0	0	2	3
Leukopenia	0	0	1	0
Catheter related infection	0	0	1	0

Responses

Two partial responses were observed in the sixteen patients who were evaluable for response. One partial response was observed at the 225 mg/m² dose level in a patient with metastatic osteosarcoma to the lung. Unfortunately, the patient had prolonged thrombocytopenia following his third course and had to be removed from protocol therapy. The second partial response was observed at the 285 mg/m² dose level in a patient with a paraspinal Ewing sarcoma. This patient received eight cycles of therapy before eventually having progressive disease.

Pharmacokinetics

Results of Day 1, Cycle 1 pharmacokinetic studies are shown in Table 4. Pharmacokinetic analyses were completed for 18 patients. The mean half-life for imetelstat was 5.3 ± 3.7 hours. Imetelstat clearance was dose-dependent. A 3-fold increase in Cmax and a 4-fold increase in AUC were observed when the dose was escalated from 225 mg/m² to 360 mg/m².

Table 4.

Summary of Imetelstat Pharmacokinetic Parameters

Dose Level		Cmax (μg/ml)	Half-life (hr)	AUC (μg/ml-hr)	Cl (ml/hr/m2)
(mg/m2)	# pts
225	7	63.7 ± 18.7	3.7 ± 1.8	307 ± 154	855 ± 298
285	6	96.7 ± 31.4	8.0 ± 5.3	614 ± 248	792 ± 602
360	5	164 ± 41.3	4.4 ± 1.5	1240 ± 395	318 ± 117

Telomerase activity in PBMCs following imetelstat treatment

The effect of imetelstat on telomerase activity in PBMC extracts was assessed using the telomeric repeat amplification protocol (TRAP). PBMC were collected prior to treatment; 6–8 hours, and 24 (± 2 hr) hours after the first imetelstat dose; prior to the imetelstat dose on Day 8; and prior to the imetelstat dose on Day 1 of Cycle 2. Samples from six patients, including three at the recommended phase 2 dose (285 mg/m²), were able to be analyzed. Althought baseline and post-treatment samples were submitted from seventeen patients, only six patients had samples that could be evaluated for telomerase inhibition. There did not appear to be any issues with assay quality. For the samples analyzed the internal controls functioned appropriately and problems appeared to be a result of poor sample quality related to shipping and handling. If telomerase activity could not be assessed at baseline, the patient was considered inevaluable for assessment of telomerase inhibition.

Telomerase inhibition in PBMCs was observed in5 of 6patients including all three that were evaluabel at the at the recommended phase 2 dose. In one of these patients telomerase inhibition was sustained through Day 8. In the other two patients, telomerase activity returned by the second dose of drug on Day 8 (for one inhibition was sustained for 6–8 hours and for the other inhibition lasted through 24 hours). At the 360 mg/m² level, two of three patients had inhibition of telomerase activity in PBMCs. For both patients, telomerase activity returned by the second dose of drug on Day 8. Figure 1 shows the telomerase inhibition results for patient #12, the patient with Ewing sarcoma who had a partial response at 285 mg/m².

Figure 1.

PBMC telomerase activity in a representative patient as assessed by the telomeric repeat amplification protocol. Lane 1 shows presence of telomerase activity pre-dose characterized by 6-bp telomeric repeat ladder TRAP products, but no activity at Day #2 or Day #8 of Cycle #1, lanes 3–4. Lane 2 represents telomerase activity 6–8 hrs after the first dose. Telomerase activity is again observed on the pre-dose sample for Cycle #2; lane 5–6, indicating that telomerase inhibition is sustained through Day #8. A and B in lanes 5–6 indicate blood samples from two different vials drawn at the same time point. This patient with paraspinal Ewing sarcoma had a partial response to therapy before having progressive disease after Cycle #8 of therapy. Lane 7, represents telomerase activity pre-dose at day 1 of cycle 3. IC is the PCR internal control; lane 8, Taq polymerase negative control; lane 9, telomerase activity negative control (lysis buffer only, CHAPS); lane 10, telomerase activity positive control (from 1000 HeLa cells).

Table 5 shows imetelstat pharmacokinetics (peak concentration and AUC) for the patients in whom telomerase inhibition could be assessed. As shown in the table, duration of telomerase inhibition did not clearly correlate with either imetelstat concentration or imetelstat AUC. However, the data are limited and insufficient to definitively evaluate the association between PK and duration of telomerase inhibition.

Table 5.

Relationship between Pharmacokinetics and Telomerase Inhibition

Patient ID #	Dose Level (mg/m2)	Cmax (μg/ml)	AUC (μg/ml-hr)	Duration of Peripheral Blood Mononuclear Cell Telomerase Inhibition1
10	285	141	816	8 hours
11	285	113	906	24 hours
12	285	115	770	8 days
15	360	199	1508	24 hours
16	360	207	1308	24 hours
18	360	169	1696	No inhibition observed

¹PBMC were collected at the following time points: 1) prior to the first dose of drug on Day 1, 2) 6–8 hours after the first dose, 3) 24 (± 2 hr) hours after the first dose, 4) prior to the imetelstat dose on Day 8, and 5) prior to the imetelstat dose on Day 1 of Cycles 2 and 3. Duration of inhibition is determined as the last time point at which telomerase activity was absent.

DISCUSSION

This study examined the toxicity and tolerable dose of imetelstat in pediatric patients with refractory or recurrent solid tumors. The maximum tolerated dose of imetelstat was 285 mg/m² intravenously over 2 hours on Days 1 and 8 of a 21-day cycle. The dose limiting toxicities observed at 360 mg/m² were myelosuppression leading to delay in therapy. In general, the hematologic and non-hematologic toxicities on this study were minor. No ≥ grade 3 non-hematologic toxicities were observed on any of the courses of therapy delivered.

Drug disposition of imetelstat was investigated on Day 1 of Cycle 1. Pharmacokinetic analyses were completed for 18 patients. The mean half-life for imetelstat was 5.3 ± 3.7 hours. Consistent with the adult pharmacokinetic data, imetelstat clearance in pediatric patients was dose-dependent. A 3-fold increase in Cmax and a 4-fold increase in AUC were observed when the dose was escalated from 225 mg/m² to 360 mg/m². The Cmax (96.7 ± 31.4 μg/ml) and AUC (614 ± 248 μg/ml•hr) at the recommended phase 2 dose (285 mg/m²) were less than have been reported for adults. In studies in adult patients with solid tumors at doses of 7.5–9.4 mg/kg (approximately 225–282 mg/m²), the Cmax and AUC were 136–190 μg/ml and 1028–1036 μg/ml•hr, respectively.(22)

Correlative biology studies show that imetelstat decreased telomerase activity in peripheral blood mononuclear cells. Telomerase inhibition was observed for 5 patients including all three at the at the recommended phase 2 dose (285 mg/m²) who had evaluable samples. In one of these patients telomerase inhibition was sustained through Day 8. In the other two patients, telomerase activity returned by the second dose of drug on Day 8. At the 360 mg/m² dose level telomerase inhibition was observed in two of three patients and activity returned by Day 8 in both patients. There was no clear predictor of the duration of telomerase inhibition. In summary, 20 children with recurrent or refractory solid tumors were enrolled in the phase 1 trial of imetelstat, and we recommend a phase 2 dose of 285 mg/m² on Days 1 and 8 of a 21-day cycle. Imetelstat pharmacokinetics were dose dependent and telomerase inhibition was observed at the recommended phase 2 dose. Two partial responses were also observed. Overall the drug was well-tolerated and the preliminary response data suggest further development of imetelstat as both a single agent or in combination studies for children. A phase 2 single agent study in pediatrics is planned.

Statement of Translational Relevance.

Telomeres, specialized structures found at the end of chromosomes, are involved in the replication and stability of the chromosome. When telomeres become critically short, cells either becomes senescent or undergo apoptosis. The enzyme telomerase plays an important role in the formation, maintenance, and renovation of telomeres. Telomerase activation is believed to be critical in cancer progression because it enables tumor cells to maintain their telomere length and avoid apoptosis. Inhibition of telomerase is an attractive new target for cancer therapy. Imetelstat is a covalently-lipidated 13-mer thiophosphoramidate oligonucleotide that acts as a potent specific inhibitor of telomerase and is a first in class agent. This study reports the first trial of imetelstat in pediatric cancer patients. Twenty subjects were enrolled in this Phase I study. Telomerase inhibition was demonstrated in 5 of 6 patients at or exceeding the phase 2 recommended dose and two confirmed partiel responses were observed.. Further development of this novel agent for pediatric cancer therapy should be considered.