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Interferon-alpha is a cytokine with demonstrated activity in patients with supratentorial gliomas, but the ideal dose and schedule of administration is unknown. Studies suggest low-dose continuous exposure is more efficacious than intermittent high doses. We performed a Phase II study of PEG-Intron® in children with DIPG, a population with dismal survival despite decades of clinical investigation. The primary objective was to compare 2-year survival to a historical cohort treated with radiation therapy alone.
Patients received weekly subcutaneous PEG-Intron® at a dose of 0.3 μg/kg beginning 2–10 weeks after completing radiation therapy until disease progression. Patients were evaluated clinically and radiographically at regular intervals. Serum and urine were assayed for biomarkers prior to each cycle. Quality of life (QOL) evaluations were administered at baseline and prior to every other cycle of therapy to parents of patients ages 6–18 years.
Thirty-two patients (median age 5.3 years, range 1.8–14.8) enrolled and received a median of 7 cycles of therapy (range 1–70+). PEG-Intron® was well tolerated and no decrease in QOL scores was noted in the subset of patients tested. The two-year survival rate was 14%, which was not significantly improved compared to our historical cohort. However, median time to progression (TTP) was 7.8 months, which favorably compares to recent trials reporting TTP of 5 months in a similar population.
Although low dose PEG-Intron® therapy did not significantly improve 2-year survival in children with DIPG compared to a historical control population, it may delay time to progression.
Diffuse intrinsic pontine gliomas (DIPG) account for up to 80% of brainstem tumors in the pediatric population1 with an estimated 300 children diagnosed in the United States each year. Because of their tenuous location, surgical resection is not possible and the mainstay of treatment is radiation therapy. The majority (~75%) of patients will initially improve clinically with radiation therapy, but tumor control is short-lived. Adjuvant chemotherapy has not significantly improved outcome. Recent Pediatric Brain Tumor Consortium (PBTC) trials2–3 have demonstrated an expected median progression-free survival (PFS) of less than 5 months in a homogeneously defined population of children with DIPG (personal communication, James Boyett). Two most recent national consortia trials (COG ACNS0126 and CCG-9941) reported mean 1 year overall survival (OS) of 40% ± 6.5% and 32% ± 6%, respectively,4 confirming the absence of progress in improving outcome and emphasizing the need for new approaches to therapy.
The interferons (IFN) are a family of glycoproteins with antiproliferative, antiviral, and immune-modulating effects.5–7 Clinical studies using alpha, beta and gamma interferons in patients with malignant brain tumors, including brainstem gliomas, have been performed with reported response rates of up to 40%.19–21 Despite the number of clinical studies performed using interferons, the ideal route of administration, schedule and dose necessary to produce maximum antitumor effects is unknown. Interferon has traditionally been administered intravenously in high doses, ≥3,000,000 U/m2, 2–3x/week, but significant side effects, including neurotoxicity, have limited its use.19 In a follow-up study of pediatric patients with brainstem glioma treated with intravenous (IV) recombinant β-interferon during hyperfractionated radiation therapy, thirteen of thirty-two (41%) patients required dose modifications due to hepatic or hematologic toxicity.22
A recent in vitro study compared high-dose administration versus frequent low-dose administration of interferon alfa-2a.13 Frequent low-dose administration produced more significant inhibition of angiogenesis-regulating genes, tumor vascularization and tumor growth compared to the higher intermittent dose schedule.13 These effects were lost at higher doses, suggesting metronomic administration of interferons may have a more robust antitumor effect. PEG-Intron® is a covalent conjugate of recombinant interferon alfa-2b with monomethoxy polyethylene glycol (PEG) and is FDA-approved for use in patients with Hepatitis C at a dose of 1 μg/kg/week. Pegylation increases the biologic half-life of the compound, enabling it to be administered once weekly, and reducing the peaks and troughs in interferon alpha-2b blood levels. Maximum serum concentrations of PEG-Intron® occur between 15–44 hours after dosing, and are sustained for up to 72 hours.23
We performed a study administering weekly dosing of PEG-Intron® as a means of establishing continuous low-dose levels of interferon in children with diffuse intrinsic pontine glioma. The dose selected was the human equivalent of the interferon alfa-2a dose with greatest observed antitumor activity used in the study by Slaton et al.13 The primary objectives were to determine its tolerability and to compare 2-year survival of children with DIPG receiving weekly low-dose PEG-Intron® after standard radiation therapy versus historical controls who received radiation therapy alone. Secondary objectives of this trial included determination of time to progression, exploration of potential biomarkers, and evaluation of quality of life.
Patients ≤ 21 years of age with a DIPG treated with radiation only and no prior chemotherapy or radiosensitizers were eligible. For this study, DIPG was defined as a diffuse intrinsic tumor on magnetic resonance imaging (MRI) with the epicenter presumed to be in the pons, a signal abnormality involving at least 50% of the pons on the T-2 weighted sequence at diagnosis, involvement of the ventral pons, and no primary exophytic component. Patients with known or suspected neurofibromatosis type 1 were excluded. Eligible patients had an ECOG performance status of ≤ 3; adequate hematological function defined as an ANC >1000/mm3, hemoglobin >8 gm/dL, and platelet count >100,000/mm3; adequate renal function defined as a normal age-adjusted creatinine or a creatinine clearance ≥ 60 mL/min/1.73 m2; and adequate hepatic function defined as total bilirubin ≤2.0 times the upper limit of normal, direct bilirubin within normal limits, and SGPT <2.5 × the upper limit of normal. Patients had to be on a stable or decreasing dose of steroids for ≥1 week prior to study entry. The study was approved by the institutional review board and continuing approval was maintained throughout the study. All patients or their legal guardians signed a document of informed consent, and verbal assent was obtained from patients when appropriate.
Treatment with PEG-Intron® was initiated 2–10 weeks after completion of radiation therapy. Each cycle of therapy consisted of 0.3 μg/kg PEG-Intron® administered subcutaneously weekly for 4 weeks. Initially, patients were instructed to receive premedication with acetaminophen or ibuprofen, but this was later omitted because of absence of PEG-Intron® side effects. Treatment with PEG-Intron® continued until unacceptable toxicity or disease progression. Disease progression was defined radiographically as the presence of new areas of tumor or ≥25% increase in tumor size, clinically as worsening neurologic symptoms despite an increase in steroids, or worsening neurologic symptoms with any increase in tumor size. Attempts were made using proton spectroscopy, FDG PET imaging, and steroid trials to distinguish radiation necrosis from tumor progression in children with worsening neurological symptoms and debatable findings on MRI.
Dosing of PEG-Intron® was held for any Grade 3 or 4 nonhematologic toxicity probably or definitely attributed to PEG-Intron®, with the exception of fever, myalgias, arthralgias, and rigors. If a toxicity did not return to ≤ Grade 1 within 14 days, PEG-Intron® was discontinued. If the toxicity returned to ≤ Grade 1 within 14 days, the patient was permitted to restart PEG-Intron® at 50% of the dose (i.e. 0.15 μg/kg once weekly) and remain at that dose. If the toxicity recurred and was attributable to PEG-Intron®, no further PEG-Intron® was given. Patients with hematologic toxicity defined as an absolute neutrophil count (ANC) < 500 for ≥ 5 consecutive days, or requirement for platelet transfusions on more than 2 days of any one cycle for platelet counts less than 50,000/μl discontinued PEG-Intron® therapy. Patients who discontinued PEG-Intron® therapy remained on study for endpoint analysis only.
Patients received a physical examination, including a detailed neurologic examination, monthly. A complete blood count, electrolytes, blood urea nitrogen, creatinine, serum total bilirubin and alanine aminotransferase were obtained at baseline and weekly during the first cycle. If no > Grade 1 laboratory toxicity was observed during the first cycle, CBC was then performed every 2 weeks on subsequent cycles, and as clinically indicated. Prothrombin time, partial thromboplastin time, calcium, phosphorous, magnesium, uric acid and urinalysis were evaluated prior to each cycle and as clinically indicated. Serum and urine were assayed for both bFGF and VEGF prior to the start of each new cycle (i.e. every 28 days). Parents of children ages 6 to 18 years with DIPG completed the Impact of Pediatric Illness (IPI) Scale24 to assess quality of life (QOL) at baseline and every other cycle. This scale measures four domains: daily activities, medical/physical status, emotional functioning, and cognitive problems. Items are rated on a 5-point Likert scale, and higher scores indicate better QOL.
Standard MRI scans to determine disease status were obtained prior to cycles 1, 2, 3, 5, 7, and continuing every other month, and when clinically indicated. Brain MRIs included proton nuclear magnetic resonance spectroscopy (NMRS), and diffusion-weighted, dynamic-enhanced and dynamic susceptibility MR sequences when possible.
Patient specimens were collected and immediately stored at -80 degrees Celsius until assayed. Samples were thawed on wet ice three hours prior to assay. Serum/urine bFGF-1 and MMP-9 ELISAs were completed as per manufacturer protocols (R&D Systems, Minneapolis MN). Samples were plated in 96-well format in duplicate and conjugated secondary antibody was added. The substrate solution (H2O2/tetramethylbenzidine) was then administered for thirty minutes and the reaction subsequently quenched with sulfuric acid. Plates were read at an absorbance of 450 nm on a Victor 3 plate reader (Perkin Elmer, Boston MA). The extrapolated absorbance was analyzed using Masterplex Readerfit ELISA software (Hitachi, Waltham MA) and the concentration determined following a 4 Parameter Logistic curve fit as per manufacturer’s recommendation. VEGF levels in serum and urine specimens were measured using an electrochemiluminescent immunoassay as per the manufacturer protocol (Meso Scale Discovery, Gaithersburg MD). Samples were assayed in 96 well plates, incubated with anti-hVEGF antibody, and read with a MSD SECTOR imager. Concentrations were determined using a 4 Parameter Logistic curve fit. Urinary biomarker values were normalized for creatinine levels obtained using the Bayer DCA 2000+ Analyzer (Bayer Healthcare, Elkhart IN) according to manufacturer’s instructions.
The primary objective of this trial was to compare survival of patients treated with radiation therapy followed by PEG-Intron® versus a historical cohort of patients who had received radiation alone. Based on a literature review from 1980–2000, median survival for children receiving radiation therapy only was approximately 11 months and 2-year survival was less then 20%.25–27 A 1-tailed exact binomial test of p=0.2 at the 0.1 significance level was used for analysis. Using a sample size of 32 patients, the power to distinguish a 2-year survival rate of 40% (H1) from the historical control rate of 20% (H0) was 88%. Overall survival was measured from the date of diagnosis to date of death. Analysis of toxicity and tolerability was descriptive.
Evaluations for the secondary objectives were planned for all patients, although neuropsychological testing was not performed if the patient was ill. Total mean parent IPI Scale scores were compared from baseline to pre-cycle 3 using repeated measures Analysis of Variance.
Patient characteristics are listed in Table 1. Thirty-two patients were enrolled and evaluable for the primary study endpoint. Median age at study entry was 5.3 (range 1.8–14.8) years; three patients were ≤ 3 years of age at diagnosis. The majority of patients began radiation within 6 weeks of becoming symptomatic; 1 patient who was diagnosed with DIPG as an incidental finding deferred radiation therapy for 5 months at which time she was symptomatic. Another patient had an abnormal MRI scan with a “lacy signal abnormality” in the pons 3.5 years prior to enrolling on study; his symptoms at that time were headaches and vertigo. Because the radiographic and clinical features were atypical for DIPG, he was not treated at the time and followed by imaging. His tumor progressed 3.5 years later both clinically and radiographically evolving to a typical radiographic appearance for DIPG. He began radiation therapy within one month of progression. Overall, the median time from end of radiation to enrollment on protocol was 38 days (range 15–64 days). Six patients had an increase in tumor size between diagnosis and their first post-radiation scan, prior to starting PEG-Intron®. Eighteen patients remained on steroids at the time of entry onto PEG-Intron®.
PEG-Intron® therapy was clinically well tolerated. Initially, parents were instructed to administer acetaminophen and ibuprofen prior to and for twenty-four hours after administration of PEG-Intron®. This was later amended to administer as needed, as most families were not administering acetaminophen or ibuprofen due to the lack of PEG-Intron® toxicity. There were no Grade 4 toxicities attributed to PEG-Intron®. Twenty Grade 3 toxicities at least possibly attributed to PEG-Intron® therapy were reported including asymptomatic laboratory abnormalities (neutropenia n=11, leukopenia n=1, lymphopenia n=2, elevated ALT n=1), fever (n=1), cranial neuropathy (n=2) and seizure (n=1). No patient discontinued PEG-Intron® for toxicity.
The overall 2-year survival rate from diagnosis was 14.3%. Characteristics of patients who survived at least 2 years from diagnosis are listed in Table 2. Two patients remain on study for endpoint analysis; one patient recently progressed after receiving more than 70 cycles of PEG-Intron®. Median time to progression and overall survival from diagnosis was 235 and 351 days, respectively. The Kaplan-Meier estimate (± standard error) of OS at 1-year was 0.4643 ± 0.0918. If the two outlying patients (i.e. those with delay in starting radiation) are excluded, median time to progression is 231 days, median OS remains 351 days, and the Kaplan-Meier estimate (± standard error) of OS at 1-year was 0.425 +/− 0.095.
Serial serum and urine samples were available for 27 patients. We used a stepwise threshold analysis to evaluate if biomarkers could be used as a marker of treatment futility. As VEGF and bFGF are two presumptive effector molecules for interferon alfa-2b therapy, we initially used a threshold of 2.2 pg/mg for normalized urinary VEGF and 6 pg/mL for serum bFGF. Using these thresholds, 17 patients had values greater than 2.2 (VEGF) and 6 (bFGF), and each of these patients progressed before 2 years. In the remaining 10 patients, using a threshold for serum MMP-9 of 5 ng/mL after the first cycle of therapy, 4 additional patients failed within 2 years. Of the remaining 6 patients, those 4 patients with a low initial VEGF and bFGF and a non-rising MMP-9 survived longer than two years.
Of the 32 children enrolled, 11 were in the age range for the IPI Scale and received a baseline QOL assessment. Of these patients, 3 were not evaluated prior to Cycle 3 due to scheduling issues and 1 discontinued PEG-Intron® due to progressive disease, therefore the pre-cycle 3 evaluations were completed on 7 patients. The mean age of these patients at baseline was 9.4 years (range = 7.5 – 12.3 years). Total mean scores improved significantly from baseline to the pre-cycle 3 assessment (baseline = 3.59 to pre-cycle 3 = 3.89; F=6.51; p = .0434), suggesting an early improvement in QOL over the first 2 months of the study. Four patients had an improvement in their mean total score (scores increased by ≥ 0.5 standard deviation), 3 showed stable scores, and none of the 7 patients’ mean total scores declined by ≥ 0.5 standard deviation.
Although interferons have a number of effects on the growth and proliferation of cells, and have demonstrated variable activity against gliomas in clinical trials, the ideal dose, schedule and type of interferon for antiglioma activity has not been identified. In this clinical trial evaluating continuous low-dose exposure of interferon alpha-2b in children with DIPG, our results indicate that PEG-Intron® administered as monotherapy after radiation therapy was not effective in significantly improving 2-year survival compared to our defined historical cohort who had received radiation only. However, in comparison to more recent trials with a similarly defined patient population, our study suggests that time to progression may be prolonged.
The historical control initially defined in this protocol was based on a literature review which spanned the time period when routine use of MRI scans was increasing and subsets of brainstem gliomas were being defined.27 While brainstem tumors are now recognized as a heterogeneous group of tumors with some subsets, such as focal or primary exophytic, having superior outcomes compared to diffuse intrinsic lesions,28 many clinical trials performed within the historical time period did not distinguish these. As a result, median and overall survival of the historical cohort may be erroneously high. Our results compare favorably to more recent clinical studies of a defined DIPG population, including ACNS 0126 with a 2 year OS of 3.6% ± 2.5%,4 and the series of recent PBTC clinical trials with estimated time to progression from diagnosis of under five months. As noted in Table 2, two patients had a somewhat atypical course in that they delayed radiation therapy after initial diagnosis. However, they met criteria for the diagnosis of DIPG and at the time of beginning radiation therapy, followed a typical course. One patient died of disease 17 months after beginning radiation therapy for DIPG. The other is alive 34 months after radiation therapy but with progressive disease first identified < 2 years from diagnosis. This suggests that, although the preradiation course was prolonged, once the patients required standard treatment for their DIPG, they followed a more typical course.
This study highlights the difficulties and limitations of performing clinical trials in children with DIPG. In addition to a limited historical cohort, a number of restrictions exist including the lack of a tissue diagnosis and absence of a standard definition of progression. The small number of patients makes it difficult to complete trials quickly and detect modest improvements in outcome. Evaluating response to therapy for patients with brain tumors, particularly DIPG, is also challenging because MRI cannot clearly distinguish tumor progression from the effects of treatment. This is made even more complicated when evaluating noncytotoxic agents, including those with an unclear or multipronged mechanism of action, such as the interferons.
The antitumor mechanism of action of interferons is not fully understood. The interferons are involved in the control of cell function and replication. The type I interferons, IFN-alpha and IFN-beta, are negative regulators of cell growth and can modify cell differentiation.8 They inhibit mitotic activity, block transition from the G0/G1 into the S phase of the cell cycle and suppress expression of receptors for certain growth factors.9–12 They down-regulate the expression of proangiogenic molecules including bFGF, IL-8, MMP-2 and MMP-9,13 and chronic systemic administration has produced regression of vascular tumors.14–16 The type I interferons also play a role in tumor immunosurveillance.17 Impaired IFN-α production has been associated with an increased risk of cancer and increased rate of tumor growth in mouse glioma models.18
No blood or tissue biomarker to date reliably predicts response to treatment or reflects response to antiangiogenic agents, although a number of potential candidates are under investigation. Evaluation of blood and urine markers rather than tissue markers are more appropriate for the DIPG population due to the inability to sample tumor tissue. VEGF, bFGF and MMPs have been shown to have prognostic value in different tumor types.29 Urinary VEGF levels have previously been shown to be predictive of outcome in cancer patients treated with radiation therapy,30 and bFGF levels in urine have correlated with disease status in cancer patients.31 Interferon alfa-2b inhibits angiogenic factor expression by decreasing production.32 An intriguing finding in our study was the identification of long-term survivors using early biomarkers. Although the number of patients was small, 4 of 6 patients with low urine VEGF, low serum bFGF and a nonrising MMP-9 survived 2 years or more, while no patient with a high urine VEGF, high serum bFGF or rising MMP-9 was a long-term survivor. Thus, a combination of biomarkers may be useful in identifying patients that may respond to interferon alfa-2b therapy.
This clinical trial demonstrates the tolerability of PEG-Intron® in this patient population. This was not unexpected, as we utilized a dose much lower than the FDA-approved dose of PEG-Intron®. Although the QOL results should be interpreted with caution due to the limited number of patients evaluated, and improved QOL scores may be, at least in part, related to symptomatic improvement from radiation therapy, a detrimental effect on QOL was not observed in any patient assessed. This is in marked contrast to prior studies of interferons which demonstrated significant toxicities, resulting in dose reduction or discontinuation.22,21,33
In conclusion, this study demonstrates that PEG-Intron®, administered to children with DIPG after radiation therapy, does not improve 2 year overall survival compared to a historical control population. However, patients on this study had prolonged time to progression compared to a contemporary, similarly defined population, with no significant adverse effect on quality of life in the small number of patients evaluated. As a monotherapy, PEG-Intron®, may not be adequate, but use in combination studies may be appropriate.
Funding: This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Conflict of Interest: The authors report no conflicts of interest.