phase i study of everolimus in pediatric patients with refractory solid tumors Phase I Study of Everolimus in Pediatric Patients With Refractory Solid Tumors

Phase I Study of Everolimus in Pediatric Patients With Refractory Solid Tumors

  1. Wayne L. Furman
  1. From the Departments of Oncology, Radiological Sciences, Biostatistics, Developmental Neurobiology, Pharmaceutical Sciences, Pathology, and Molecular Pharmacology, St Jude Children's Research Hospital; and Department of Pediatrics, The University of Tennessee School of Medicine, Memphis, TN
  1. Address reprint requests to Maryam Fouladi, MD, Department of Oncology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794; e-mail: maryam.fouladi{at}stjude.org
 
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Abstract

Purpose To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic and pharmacodynamic properties of the mammalian target of rapamycin (mTOR) inhibitor, everolimus, in children with refractory or recurrent solid tumors.

Patients and Methods Everolimus was administered orally at a daily dose of 2.1, 3, 5, or 6.5 mg/m2 in cohorts of three to six patients per dosage level. Pharmacokinetic and pharmacodynamic studies were performed during the first course. The phosphorylation status of various components of the mTOR signal pathway was assessed in peripheral-blood mononuclear cells (PBMCs) isolated from treated patients.

Conclusion Continuous, orally administered everolimus is well tolerated in children with recurrent or refractory solid tumors and demonstrates similar pharmacokinetic properties to those observed in adults. Everolimus significantly inhibits the mTOR signaling pathway in children at the MTD. The recommended phase II dose in children with solid tumors is 5 mg/m2.

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INTRODUCTION

The cell signal pathways that activate the mammalian target of rapamycin (mTOR) are altered in many human cancers.1-8 mTOR is a ubiquitous serine threonine kinase involved in the regulation of cell cycle, angiogenesis, and apoptosis.9-11 This protein is incorporated into the following two distinct multiprotein complexes: mTOR complex (mTORC) 1, which regulates growth through effectors such as S6K1 and 4E-binding protein 1, and mTORC2, which appears to regulate AKT/PKB by phosphorylating AKTSer473, thereby impacting cell survival.12

Rapamycin and its analog, everolimus, are small-molecule inhibitors of mTOR. They form a complex with FK506-binding protein 12 and mTOR, inhibiting mTOR and leading to antiproliferative effects, including G1-phase cell cycle arrest13,14 and apoptosis.14 Rapamycin derivatives can block activation of AKT signaling by inhibiting the formation of mTORC215,16 and can inhibit angiogenesis.17

Everolimus has potent in vitro activity against many human cancer cell lines and xenograft models.13,14,18-27 Rhabdomyosarcoma, neuroblastoma, medulloblastoma/primitive neuroectodermal tumor (PNET), and pediatric glioblastoma cell lines are also sensitive to rapamycin analogs (concentration required for 50% inhibition from 0.37 to 4,680 ng/mL),28,29 with activity also reported in xenograft models.30,31 Furthermore, rapamycin analogs are highly lipophilic and can cross the blood-brain barrier.30

Everolimus is undergoing adult phase I and II trials. In adults with recurrent hematologic malignancies, everolimus was well tolerated at 5 to 10 mg orally daily, with no dose-limiting toxicities (DLTs) reported. Toxicities included hyperglycemia, hypophosphatemia, fatigue, anorexia, and diarrhea. Analysis of patients' peripheral-blood mononuclear cells (PBMCs) identified significant inhibition of phosphorylation of mTOR targets, including eukaryotic initiation factor 4E-binding protein 1, AKT, and p70 S6 kinase.32

We report the results of a phase I trial of everolimus in children with recurrent or refractory solid tumors. The primary objectives were to estimate the maximum-tolerated dose (MTD) and DLTs of everolimus administered orally daily. The secondary objectives were to assess the biologic activity of everolimus by measuring targets of mTOR in PBMC, to characterize everolimus pharmacokinetics in children, and to provide preliminarily data on antitumor activity.

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PATIENTS AND METHODS

Patient Eligibility

Patients had to be ≥ 3 and younger than 22 years old with a histologically verified solid tumor refractory to conventional therapy; a Lansky or Karnofsky performance score ≥ 50; and a life expectancy of ≥ 8 weeks. Patients had to have a body-surface area of more than 1.0, and 0.8, and 0.5 m2 for dose level 0, and 1, and higher, respectively. Patients must have recovered from the acute toxic effects of prior therapy and must not have received growth factors within 1 week of study entry, myelosuppressive chemotherapy within 3 weeks (4 weeks if prior nitrosourea, temozolomide, or mitomycin), craniospinal or total-body irradiation within 3 months, local palliative radiotherapy within 8 weeks to the primary tumor, or focal irradiation to symptomatic metastatic sites within 2 weeks. Patients on cyclosporine or tacrolimus, H2 antagonists or proton pump inhibitors (unless on corticosteroids), enzyme-inducing anticonvulsants, or other potent cytochrome P450 3A4 inhibitors were excluded, as were pregnant or lactating women or patients with uncontrolled infections. Corticosteroids, at stable or decreasing dose for ≥ 1 week before start of therapy, were only permitted for the treatment of increased intracranial pressure or cord compression. Other requirements included adequate bone marrow function (peripheral absolute neutrophil count ≥ 750/μL, platelet count ≥ 75,000/μL [transfusion independent], and hemoglobin ≥ 8.0 g/dL), adequate renal function (age-adjusted normal serum creatinine or glomerular filtration rate ≥ 70 mL/min/1.73 m2), adequate liver function (total bilirubin ≤ 1.5× institutional upper limit of normal for age and ALT ≤ 3× institutional upper limit of normal for age), adequate cardiac function (shortening fraction ≥ 27% by echocardiogram or left ventricular ejection fraction > 50% by gated radionuclide study, normal ECG), stable neurologic deficits for ≥ 1 week, adequate pulmonary function, and normal serum sodium, calcium, magnesium, and potassium. Informed consent was obtained from patients, parents, or guardians, and assent was obtained as appropriate, at the time of protocol enrollment. The protocol was approved by the institutional review board.

Drug Administration and Study Design

Everolimus was supplied by Novartis (Emeryville, CA) as 2.5- and 5-mg tablets and was administered orally daily (one course = 28 days). A dosing nomogram based on body-surface area and dose level was used to prescribe everolimus for patients to minimize interpatient dosing variability. The maximum deviation from prescribed dose was 24%. The starting everolimus dosage was 3 mg/m2 (adult recommended dose is 5 to 10 mg daily).

Readily reversible grade 3 and 4 DLTs of hypokalemia and hypophosphatemia in platinum-pretreated patients led to dosage de-escalation to 2.1 mg/m2. The definition of DLT was amended to exclude grade 3 or 4 electrolyte abnormalities that resolved to ≤ grade 2 within 7 days of interrupting treatment, allowing further dosage escalation in 30% increments.

A minimum of three assessable patients were treated at each dose level. If one of three patients at a given dose level experienced a DLT, three more patients were accrued at the same dose level. If two or more patients experienced DLT, then the MTD was exceeded, and three more patients were treated at the next lower dose level. The MTD was defined as the dose level at which, at most, one patient of six experienced a DLT with at least two of three to six patients experiencing a DLT at the next higher level. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (version 3.0).

Hematologic DLT was defined as grade 4 neutropenia or grade 3 or 4 thrombocytopenia related to everolimus. Nonhematologic DLT was defined as grade 3 or 4 nonhematologic toxicity with the specific exclusions of grade 3 nausea and vomiting controlled with adequate antiemetic prophylaxis, grade 3 transaminase elevations that returned to baseline within 7 days of interrupting treatment, grade 3 hyperlipidemia, grade 3 fever or infection, and grade 3 or 4 electrolyte abnormalities that resolved to ≤ grade 2 within 7 days of interrupting treatment.

Pretreatment evaluations included a history, physical examination, performance status, disease evaluation, CBC, electrolytes, renal and liver function tests, cholesterol, high-density lipoprotein to low-density lipoprotein ratio, triglycerides, serum protein, and albumin. CBCs were obtained twice weekly during course 1, weekly during course 2, and before each subsequent course. Histories, physical examinations, and serum chemistries were obtained weekly in course 1 and before each subsequent course.

Disease evaluations were obtained at baseline, after course 2, and at every third course thereafter. Tumor response was reported using the Response Evaluation Criteria in Solid Tumors.33 For CNS malignancies, response was defined as follows: complete response was the disappearance of all measurable lesions on magnetic resonance imaging (MRI); partial response was ≥ 50% reduction in the sum of the product of the maximum perpendicular diameters of all measurable lesions on MRI, with decreasing corticosteroid doses and stable or improving neurologic examination maintained for ≥ 4 weeks; and progressive disease was worsening neurologic status or more than 25% increase in the product of the maximum perpendicular diameters of any lesions, appearance of new lesions, or increasing corticosteroid doses. Patients were considered to have stable disease (SD) if the MRI response did not meet the criteria for the other categories and the patient had stable neurologic examination and corticosteroid dose. All imaging was reviewed by the study radiologist (F.L.).

Pharmacokinetics Studies

Pharmacokinetic studies were performed on day 1 of course 1 in consenting patients. Whole-blood samples (3 mL) were collected in an EDTA tube before everolimus administration and at 0.5, 1, 1.5, 2, 4, 6, 8, and 24 hours after administration. Samples were processed, and everolimus concentrations were analyzed using a modification of a previously published high performance liquid chromatography–tandem mass spectrometry method that was implemented and validated in our laboratory.34 Both intraday and interday quality control samples (5.0 ng/mL) showed a coefficient of variation of less than 6%. We noted a higher variability of 10% (coefficient of variation) for concentrations of 1 ng/mL.

A two-compartment model was fit to the everolimus whole-blood concentration-time data after oral administration from all individuals simultaneously via nonlinear mixed-effects modeling as implemented by NONMEM V (Globomax, LLC, Hanover, CT) using first-order conditional estimation with interaction. The pharmacokinetic parameters for each patient were determined using the posthoc option in NONMEM. A log-normal distribution was assumed for each of the model parameters (elimination rate constant, ke; volume of distribution, V; intercompartmental rate constants, kcp and kpc; absorption rate constant, ka; and bioavailability, F); therefore, for individual i, the parameters were described by pari = parpopeηI, where parpop was the population estimate of the parameter and ηi ∈ N(0, σ) described the variation of individual i from the population average. The residual error of the model was described by y = f(1 + ε1) +ε2, where y was the observed concentration, f was the predicted concentration, ε1 ∈ N(0, ω1) described the relative error, and ε2 ∈ N(0, ω2) described the absolute error. For each patient, the area under the concentration-time curve from zero to infinity (AUC0→∞) was calculated by integration of the simulated concentration-time data from model estimates.

Immunohistochemistry

We analyzed the baseline phosphorylation of S6Ser235/236 and AKTSer473 in archival pretreatment tumor samples using standard immunohistochemical (IHC) techniques.35 IHC was performed on 5-μm thick sections of formalin-fixed, paraffin-embedded tumor samples. IHC controls included sections of Daoy.V and Daoy.2 human medulloblastoma xenografts that express low and high levels of phosphorylated S6Ser235/236 and AKTSer473, respectively.36 To remove observer bias, IHC staining of tumors was scored blind to the diagnosis and treatment response using ImageJ software (National Institutes of Health, Bethesda, MD) analysis as described previously.37 The IHC score provides a measure of the mean percentage of immunopositivity that is detected in each ×200 field.

Western Blotting

Whole blood (5 mL) was obtained from consenting patients before everolimus administration and on treatment days 15, 28, and 62. PBMCs were isolated from each sample using the Ficoll reagent. Total protein was then isolated from each PBMC pellet and stored at −80°C until analyzed. The levels of S6Ser235/236 and AKTSer473 expression in PBMC isolates at each time point were determined using standard Western blotting techniques38 and recorded relative to the total level of S6 and AKT protein, respectively. Actin was used as a loading and transfer control in each Western blot analysis. All antibodies were from Cell Signaling Technology (Danvers, MA).

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RESULTS

Patient Characteristics

Twenty-six patients were enrolled onto the study; one patient was declared ineligible because he received radiotherapy within 6 weeks before starting everolimus. Table 1 lists the characteristics of the 25 eligible patients. Eighteen of the remaining 25 patients were fully assessable for DLT determination. Among the seven patients who were not, one patient experienced virally induced prolonged neutropenia, one patient never received everolimus because of deteriorating liver function, and five patients failed to complete course 1 because of consent withdrawal (n = 2), noncompliance (n = 1), or progressive disease (n = 2). The median number of courses was two (range, zero to 14 courses).

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Table 1.

Characteristics of Eligible Patients (n = 25)

Toxicity

The observed DLTs are listed in Table 2. DLTs at 3 mg/m2 included reversible hypokalemia (n = 1) and hypophosphatemia (n = 2). Dose de-escalation to 2.1 mg/m2 led to no DLTs in three assessable patients. Three more assessable patients were enrolled at 3 mg/m2 and 5 mg/m2, each with no DLTs. At 6.5 mg/m2, the DLTs included grade 3 elevation of ALT (n = 1), mucositis (n = 1), and diarrhea (n = 1). Thus, three more patients were enrolled at 5 mg/m2, with no observed DLTs, making 5 mg/m2 the recommended phase II dose. Table 3 lists the grade 3 and grade 4 adverse events at least possibly attributable to everolimus in 24 patients who received drug.

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Table 2.

DLT Summary (course 1)

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Table 3.

Grade 3 and 4 Toxicities Attributable to Everolimus by Dose Level in 24 Pediatric Patients With Refractory Solid Tumors

Responses

No objective response was reported. Prolonged SD was observed in one patient each with gliomatosis cerebri (low-grade glioma, 14 courses), osteosarcoma (eight courses), brainstem glioma (five courses), peripheral PNET (five courses), anaplastic astrocytoma (four courses), and ependymoma (four courses).

Pharmacokinetics

Nineteen patients consented to pharmacokinetic studies; one patient was excluded because of unexplainable variations in everolimus concentrations. Although whole-blood everolimus concentrations between 1 and 5 ng/mL had greater variability than concentrations greater than 5 ng/mL, we used those values and assigned a higher relative error in the NONMEM analysis.

The pharmacokinetic data were best represented by a two-compartment model (Fig 1). Population estimates for clearance (Cl)/F, V/F, and ka were 12.4 L/h/m2, 45.5 L, and 1.66 h−1, respectively. Interindividual variability in Cl/F, V/F, and ka was 47%, 46%, and 45% as determined by the percent coefficient of variation, respectively. Median apparent Cls for the 2.1, 3, 5, and 6.5 mg/m2 dosage levels were 12.1 L/h/m2 (range, 9.1 to 24.2 L/h/m2), 9.3 L/h/m2 (range, 4.3 to 28.5 L/h/m2), 15.2 L/h/m2 (range, 14.4 to 16.0 L/h/m2), and 16.3 L/h/m2 (range, 13.9 to 18.7 L/h/m2), respectively. Table 4 lists the compartmental and noncompartmental pharmacokinetic results for each dosage level.

Fig 1.
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Fig 1.

Observed everolimus concentrations for all patients. Open circles (○) represent actual concentrations, and the solid line (——) is the best-fit curve from the population pharmacokinetic analysis.

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Table 4.

Summary of Everolimus Pharmacokinetic Parameters

IHC Analysis of mTOR Signaling Activity in Tumor Samples

Archival pretreatment tumor samples were available from 14 patients. Thirteen samples displayed S6Ser235/236 and AKTSer473 immunoreactivity in an average of 33% (range, 10% to 90%) and 48% (range, 10% to 100%) of tumor cells, respectively. One tumor was negative for both phosphorylated proteins. Three pretreatment primary tumors (gliomatosis cerebri, osteosarcoma, and peripheral PNET) from patients who received ≥ five courses of everolimus contained more AKTSer473 immunoreactive cells (76%; range, 60% to 100%) than 10 tumors from patients receiving ≤ four treatment courses (39%; range, 10% to 80%), suggesting that everolimus might impede the growth of tumors that develop with high mTOR pathway activity. However, the small numbers involved in this study precluded formal statistical evaluation. No relationship was observed between the level of S6Ser235/236 immunoreactivity in pretreatment tumor samples and patients' duration of therapy.

Western Blot Analysis of mTOR Pathway Activity in PBMCs

PBMC samples were available from 19 patients. These included 16 pairs of pretreatment and on-therapy samples in patients treated at the dose levels of 2.1 (n = 5), 3 (n = 7), 5 (n = 3), and 6.5 mg/m2 (n = 1). Both total AKT and phosphorylated AKTS473 proteins were detected readily in PBMC by Western blot analysis (Fig 2A). Increases in everolimus dose were associated with a stepwise decrease in AKTS473 phosphorylation in PBMCs; 14 days of therapy with everolimus 5 mg/m2 inhibited significantly the phosphorylation of AKTS473 compared with that observed in pretreatment PBMCs (P < .01; Fig 2B). Three or more PBMC samples drawn during course 1 of therapy were available from patients receiving 2.1 (n = 1), 3 (n = 3), and 5 mg/m2 (n = 2) doses (Fig 2C). These data confirmed that doses of 3 to 5 mg/m2 of everolimus, corresponding to everolimus AUC of ≥ 200 ng/mL·h, were most effective at decreasing AKTS473 phosphorylation in PBMCs over time.

Fig 2.
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Fig 2.

Western blot analysis of total and phosphorylated AKT in peripheral-blood mononuclear cells (PBMCs). (A) PBMC analysis in three patients before (0) and 14 and 28 days after start of everolimus treatment at the indicated dose. (B) Average relative phosphorylation of AKTS473 in patients' PBMCs treated with the indicated everolimus dose at day 14. Pretreatment (0) levels are included for comparison. **P = .005. (C) Fold change in relative phosphorylation of AKTS473 in patient PBMCs treated with the indicated dose of everolimus by time on therapy.

In contrast, both total S6 and phosphorylated S6Ser235/236 levels were detected less readily in patients' PBMCs. Detectable levels of S6 proteins were identified in only eight pairs of pretreatment and on-therapy samples at 2.1 (n = 3), 3.0 (n = 3), 5.0 (n = 1), and 6.5 mg/m2 (n = 1). Fourteen days of treatment with 2.1 or 3 mg/m2 everolimus resulted in a nonsignificant decrease in S6Ser235/236 phosphorylation on PBMCs (data not shown). Total S6 and phosphorylated S6Ser235/236 proteins were undetectable in the remaining PBMCs, precluding further assessment.

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DISCUSSION

This pediatric phase I trial establishes the MTD of everolimus as 5 mg/m2 orally daily. The DLTs included diarrhea, mucositis, elevated ALT, hypokalemia, and hypophosphatemia, similar to adult studies.32 Although no objective responses were reported, six patients experienced prolonged SD (four to 14 courses).

Everolimus pharmacokinetics in children are comparable with those in adults.39 Everolimus was absorbed rapidly, with maximum concentrations achieved as early as 30 minutes after administration. The maximum everolimus whole-blood concentration and AUC at each dose level were variable but increased with dose. Interpatient variability was significant, with an approximate seven-fold variation in apparent oral clearance (4.3 to 28.5 L/h/m2). Although a three-fold range in dosages were evaluated in this trial, the wide interpatient variability in apparent oral clearance translated into a six-fold variability in everolimus systemic exposure as measured by area under the whole-blood concentration-time curve.

Phosphorylation of AKTS473 was inhibited significantly in PBMCs of children treated with everolimus 5 mg/m2. The rictor/mTOR protein complex mTORC2 phosphorylates AKT at the hydrophobic Ser473 site and is essential for AKT activity.12 Until recently, the mTORC2 complex was thought to be insensitive to inhibition by rapamycin and its derivatives. However, prolonged inhibition of mTOR by rapamycin has been shown to impair mTORC2 assembly and AKT activation in some cell types including acute myeloid leukemia cells.15,16 Indeed, daily doses of 5 or 10 mg inhibited significantly AKTS473 phosphorylation in PBMCs of six of eight acute myeloid leukemia patients,16 with significant decreases in expression of downstream target genes reported only in samples demonstrating inhibition of AKT phosphorylation.16

In contrast to their effect on normal and malignant peripheral-blood cells, inhibitors of mTOR have been shown to increase phosphorylation of AKT in adult solid tumor cells via an insulin growth factor signal–dependent mechanism.40,41 This feedback system has been suggested to mediate resistance of cancer cells to mTOR inhibitors. It remains to be determined whether similar feedback systems operate in pediatric tumor cells. However, pretreatment patient tumor samples taken from patients in the current study who received five or more courses of everolimus expressed high levels of phosphorylated AKTS473, arguing that AKT signaling per se does not mediate resistance in these pediatric tumors. Future studies will be required to determine whether tumors with higher levels of mTOR signaling are more sensitive to everolimus therapy. Nevertheless, our data indicate that phosphorylation of AKT may provide a surrogate marker to assess mTOR signal inhibition. Although preclinical and clinical studies have shown inhibition of S6S235/236 to be a reliable biomarker of mTOR blockade by rapamycin analogs in surrogate and tumor tissues,42 we found it difficult to detect reproducibly total or phosphorylated levels of S6 in pediatric PBMCs. We identified detectable levels of p70S6K in only eight pairs of pretreatment and on-treatment PBMC samples. Although we did observe inhibition of S6S235/236 with treatment, this protein proved to be a less reliable target of mTOR activity than AKT in children treated with everolimus. Finally, we observed the highest expression levels of phosphorylated AKTS473 in pretreatment tumor samples from patients who received ≥ five courses of everolimus. Future studies will be required to determine whether tumors with higher levels of mTOR signaling are more sensitive to everolimus therapy.

Given the minimal toxicity of everolimus and its additive or synergistic preclinical activity in combination with vascular endothelial growth factor receptor and ErbB inhibitors26 and cytotoxic agents such as cisplatin,24 there is considerable interest in its further development. Future pediatric trials include combinations with antiangiogenic agents or ErbB inhibitors in recurrent solid tumors and CNS malignancies.

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AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: None Consultant or Advisory Role: None Stock Ownership: None Honoraria: None Research Funding: Maryam Fouladi, Novartis Pharmaceuticals; Kristen Molina, Novartis Pharmaceuticals; Sheri L. Spunt, Wyeth Inc Expert Testimony: None Other Remuneration: None

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AUTHOR CONTRIBUTIONS

Conception and design: Maryam Fouladi, Fred Laningham, Jianrong Wu, Sheri L. Spunt, Clinton F. Stewart, Peter J. Houghton, Richard J. Gilbertson, Wayne L. Furman

Provision of study materials or patients: Maryam Fouladi, Alberto Broniscer, Sheri L. Spunt

Collection and assembly of data: Maryam Fouladi, Fred Laningham, Melinda A. O'Shaughnessy, Kristen Molina, Clinton F. Stewart

Data analysis and interpretation: Maryam Fouladi, Fred Laningham, Jianrong Wu, Melinda A. O'Shaughnessy, Kristen Molina, Inga Luckett, Clinton F. Stewart, Richard J. Gilbertson, Wayne L. Furman

Manuscript writing: Maryam Fouladi, Melinda A. O'Shaughnessy, Clinton F. Stewart, Richard J. Gilbertson

Final approval of manuscript: Maryam Fouladi, Fred Laningham, Jianrong Wu, Melinda A. O'Shaughnessy, Kristen Molina, Alberto Broniscer, Sheri L. Spunt, Inga Luckett, Clinton F. Stewart, Peter J. Houghton, Richard J. Gilbertson, Wayne L. Furman

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Footnotes

  • Supported by Grants No. P30 CA21765 and P01 CA23099 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities.

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

  • Received February 21, 2007.
  • Accepted July 26, 2007.
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  1. doi: 10.1200/JCO.2007.11.4017 JCO vol. 25 no. 30 4806-4812
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