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05-2215 860.868

Phase 1Trial of Gefitinib Plus Sirolimus in Adults with RecurrentMalignant Glioma David A. Reardon,2,3 Jennifer A. Quinn,2,6 James J. Vredenburgh,2,6 Sridharan Gururangan,2,3Allan H. Friedman,2 Annick Desjardins,6 Sith Sathornsumetee,6 James E. Herndon II,7 Jeannette M. Dowell,7Roger E. McLendon,4 James M. Provenzale,5 John H. Sampson,2 Robert P. Smith,1Alan J. Swaisland,1Judith S. Ochs,1Peggy Lyons,2 Sandy Tourt-Uhlig,2 Darell D. Bigner,4 Henry S. Friedman,2,3and Jeremy N. Rich2,6 Purpose: To determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) ofgefitinib, a receptor tyrosine kinase inhibitor of the epidermal growth factor receptor, plussirolimus, an inhibitor of the mammalian target of rapamycin, among patients with recurrentmalignant glioma.
Patients and Methods: Gefitinib and sirolimus were administered on a continuous daily dosingschedule at dose levels that were escalated in successive cohorts of malignant glioma patients atany recurrence who were stratified based on concurrent use of CYP3A-inducing anticonvulsants[enzyme-inducing antiepilepticdrugs, (EIAED)]. Pharmacokineticand archival tumor biomarkerdata were also assessed.
Results: Thirty-four patients with progressive disease after prior radiation therapy and chemo-therapy were enrolled, including 29 (85%) with glioblastoma multiforme and 5 (15%) with ana-plasticglioma. The MTD was 500 mg of gefitinib plus 5 mg of sirolimus for patients not onEIAEDs and 1,000 mg of gefitinib plus 10 mg of sirolimus for patients on EIAEDs. DLTs includedmucositis, diarrhea, rash, thrombocytopenia, and hypertriglyceridemia. Gefitinib exposure wasnot affected by sirolimus administration but was significantly lowered by concurrent EIAED use.
Two patients (6%) achieved a partial radiographic response, and 13 patients (38%) achievedstable disease.
Conclusion:We show that gefitinib plus sirolimus can be safely coadministered on a continuous,daily dosing schedule, and established the recommended dose level of these agents incombination for future phase 2 clinical trials.
Traditional cytotoxic therapies, including external beam Signal transduction pathways, associated with tumor cell radiotherapy (X-ray therapy) and chemotherapy, provide a proliferation, migration, angiogenesis, and survival, provide modest survival advantage for some patients with newly multiple potential therapeutic targets currently being evaluated diagnosed glioblastoma multiforme (1). Salvage therapies are in oncology. Aberrant signaling of the phosphatidylinositol 3V- ineffective (2), and nearly all glioblastoma multiforme patients kinase (PI3K) pathway occurs frequently in glioblastoma die within 1 to 2 years of diagnosis. Innovative, more effective multiforme (3) and is associated with poor response to treatments are desperately needed for this patient population.
conventional cytotoxic therapy (4). Several molecular mecha-nisms have been linked to PI3K pathway signaling, includingactivation of upstream growth factor receptors, such as theepidermal growth factor receptor (EGFR), or loss of function Authors’ Affiliations: 1AstraZeneca Pharmaceuticals, Wilmington, Delaware; of the PTEN tumor suppressor gene, which normally Departments of 2Surgery, 3Pediatrics, 4Pathology, 5Radiology, 6Medicine, and antagonizes PI3K (5). We recently reported results of a clinical 7Cancer Center Biostatistics, Duke University Medical Center, Durham, North trial with gefitinib, a novel low molecular weight, EGFR CarolinaReceived 10/10/05; revised 11/6/05; accepted 11/11/05.
tyrosine kinase inhibitor (TKI), in recurrent glioblastoma Grant support: NIH grants NS20023 and CA11898, NIH/General Clinical multiforme patients. Although 9 of 53 patients (17%) Research Center Program/National Center for Research Resources grant MO1 RR remained progression-free for at least 6 months, the majority 30, and National Cancer Institute Specialized Programs of Research Excellence of patients suffered early disease recurrence (6). Similar, modest antitumor activity has recently been reported among The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance recurrent glioblastoma multiforme patients treated with with 18 U.S.C. Section 1734 solely to indicate this fact.
erlotinib, another EGFR TKI (7). Several possible factors may Requests for reprints: David A. Reardon, The Preston Robert Tisch Brain Tumor limit the clinical benefit associated with EGFR TKIs, including Center at Duke, Duke University Medical Center, Box 3624, Durham, NC 27710.
compensatory activation of either downstream pathway Phone: 919-668-2650; Fax: 919-668-2485; E-mail: reard003@mc.duke.edu.
components or alternative mitogenic/survival pathways, as F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-2215 well as molecular resistance mechanisms (8).
Clin Cancer Res 2006;12(3) February 1, 2006 Gefitinib Plus Sirolimus for Malignant Glioma We recently showed in preclinical studies that the antitumor activity of an EGFR TKI can be enhanced by combination with Gefitinib and sirolimus were orally administered on a continuous an inhibitor of the mammalian target of rapamycin (mTOR; daily dosing schedule of 28-day cycles (Table 1). For all patients except ref. 9). mTOR, a downstream target of the PI3K pathway, is a for those who underwent pharmacokinetic sampling, a loading dose of central regulator of several essential cellular processes in both commercially available sirolimus was administered on the first day of normal and neoplastic cells including nutrient metabolism, cycle 1 followed by a continuous daily maintenance dose. Gefitinib,provided by AstraZeneca Pharmaceuticals (Wilmington, DE), was taken cell cycle progression, and protein translation (10, 11).
concurrently with sirolimus. For patients who underwent pharmaco- Although the clinical benefit of mTOR inhibitors for malig- kinetic sampling, gefitinib was administered alone for days 1 to 7 of nant glioma patients seems modest (12, 13), the antitumor cycle 1. On day 8 of cycle 1, a loading dose of sirolimus was activity of mTOR antagonists is enhanced by loss of PTEN administered. Thereafter, gefitinib and sirolimus were administered (14), which occurs commonly in glioblastoma multiforme concurrently each day. Patients received up to 12 cycles unless (15 – 18). Thus, possible mechanisms of EGFR TKI resistance unacceptable toxicity or tumor progression occurred.
may be preferentially targeted by mTOR antagonists. To extend Gefitinib metabolism is significantly enhanced by concurrent use of this hypothesis to the development of a novel therapeutic CYP3A-inducing antiepileptic drugs (EIAED), including phenytoin, approach for malignant glioma patients, we conducted the carbamazepine, phenobarbital, oxcarbazapine, and primidone (19).
current phase I study to determine the maximum tolerated Therefore, patients were accrued independently into two separate strata:stratum A, patients not on EIAEDs; stratum B, patients on EIAEDs. A dose (MTD) of gefitinib plus sirolimus, an mTOR antagonist, ‘‘3+3’’ phase I dose escalation design was employed to determine the in patients with recurrent malignant glioma. Our report MTD for each stratum. Intrapatient dose escalation was not permitted.
describes the first study of a combinatorial regimen of The dose level was escalated in successive cohorts of three patients as long molecularly targeted agents in the treatment of recurrent as DLT did not occur. If one instance of DLT was observed among the malignant glioma patients and specifically includes a combi- initial three evaluable patients, three additional patients were treated at natorial regimen designed to simultaneously inhibit key that dose level. Dose escalation continued as long as no episodes of DLT upstream and downstream mediators of PI3K signaling.
occurred in the additional three patients. If two instances of DLT wereobserved at a dose level, the MTD was surpassed and a total of six patientswere treated at the previous level. The MTD was defined as the highest dose causing DLT in no more than one of six patients.
DLT was defined as any of the following toxicities that occurred during the first cycle of therapy: grade 4 thrombocytopenia or The primary objective was to define the MTD and dose-limiting neutropenia lasting >4 days, grade z3 nonhematologic toxicities felt toxicity (DLT) of gefitinib plus sirolimus in adults with recurrent to be related to the study regimen excluding grade z3 nausea or emesis malignant glioma. Secondary objectives included to further define the for which inadequate medical therapy was administered, and >14 day toxicity of this regimen, to obtain pharmacokinetic data, and to delay in treatment due to related toxicity. Toxicities were graded evaluate for antitumor activity relative to clinical, archival tumor according to the National Cancer Institute’s Common Toxicity Criteria biomarker, and pharmacokinetic measures.
version 3.0 and classified as related to the study regimen unless theywere attributable to either underlying tumor progression, a concurrent medical condition or a concomitant medication.
Patients were required to have histologically confirmed malignant Before each cycle, patients underwent a physical examination and glioma (glioblastoma multiforme, gliosarcoma, anaplastic astrocyto- full chemistry panel, including fasting cholesterol and triglycerides. A ma, anaplastic oligodendroglioma, or anaplastic oligoastrocytoma) complete blood count with differential was obtained weekly. In that was radiographically progressive following prior radiation or addition, before cycle 1, a urinalysis was obtained in all patients, and chemotherapy. Additional enrollment criteria included age at least 18 h-human chorionic gonadotropin was obtained in women with years, Karnofsky performance status z70%, stable corticosteroid dose for at least 1 week before therapy initiation, hematocrit >29%; absolute Response evaluation was done before each treatment cycle.
neutrophil count >1,000 cells/AL; platelet count >100,000 cells/AL, Determination of overall response was based on radiographic change serum creatinine and bilirubin <1.5 times the institutional upper limit in tumor size as revealed by computed tomography or magnetic of normal, serum aspartate aminotransferase <2.5 times the institu-tional upper limit of normal, and carbon monoxide diffusing capacityof >75% predicted. Patients were required to be at least 3 weeks from prior surgical resection and to have recovered from all toxicitiesassociated with any prior therapy. All patients were informed of the investigational nature of the study and provided informed consent as approved by the Duke University Medical Center Institutional Review Patients were excluded for any of the following: more that three prior episodes of progressive disease, pregnancy or nursing, refusal to use effective contraception if of reproductive potential, progressivedisease following prior treatment directed against either EGFR or mTOR, and acute infection requiring i.v. antibiotics. In addition, patients were not eligible if they received prior stereotactic radiosurgery, radiation implants, or radiolabeled monoclonal antibody therapy, due to the difficulty distinguishing progressive tumor from radionecrosis onmagnetic resonance imaging following such therapies. However, *Stratum A: patients not on EIAEDs (phenytoin, phenobarbitol, carbamaze- patients who had received these therapies were eligible if they had pine, oxcarbazepine, and primidone).
either biopsy confirmation of recurrent tumor, or if they had a new or progressive distant lesion on magnetic resonance imaging.
Clin Cancer Res 2006;12(3) February 1, 2006 resonance imaging and clinical criteria, including steroid requirement Corp., Mountain View, CA). Total body clearance of drug from and neurologic examination. Complete response was defined as the plasma at steady state after an oral dose (CLss/F) was calculated as disappearance of all enhancing or nonenhancing tumor from baseline on consecutive scans at least 6 weeks apart, with the patient not The paired t test was used to compare gefitinib alone AUCss (day 7) receiving corticosteroids and neurologically stable or improved. Partial to that with sirolimus (day 10) for each stratum and for each dose level.
response was defined as z50% reduction from baseline in the size A two-sample t test was used to compare dose-normalized, gefitinib (measured as the product of the largest perpendicular diameters) of AUCss from day 7 between patients on strata A and B.
enhancing tumor maintained for at least 6 weeks, use of a stable or A trough serum sirolimus level was measured after day 10 of cycle reduced corticosteroid dose, and stable or improved neurologic exam.
1. Two-way ANOVA was used in a generalized linear model Progressive disease was defined as >25% increase in size of enhancing framework to examine the effect of dose and strata on blood levels or nonenhancing tumor or any new tumor on magnetic resonance of sirolimus. This analytic approach assumed measurement errors to imaging scan or neurologic worsening of the patient without a be normally distributed, and repeated measures of sirolimus levels documented nonneurologic etiology while on a stable or increased corticosteroid dose. Stable disease was defined as any other status notmeeting the criteria for complete response, partial response, and progressive disease that was observable for more than one course of Archival tumor samples from either initial diagnosis or after prior therapy were analyzed for phospho-p44/42 mitogen-activated protein Time to progression and overall survival, measured from the date kinase (p-MAPK), p-S6 ribosomal protein, p-AKT, PTEN, and EGFR cycle 1 began, were analyzed by the Kaplan-Meier method, including using immunohistochemistry reagents and methods as described 95% confidence intervals (95% CI; refs. 20, 21).
below. Similarly, archival tumor samples were analyzed by fluorescencein situ hybridization (FISH) for EGFR and PTEN DNA locus copy Dose modification and retreatment criteria number using reagents and methods as described below. Primary The criteria for retreatment consisted of the following: absolute antibodies for immunohistochemical staining included rabbit mono- neutrophil count > 1,000 cells/AL; platelets >100,000 cells/Al; serum clonal p-MAPK (Thr202/Tyr204, clone E10), rabbit polyclonal p-S6 aspartate aminotransferase, total bilirubin, and creatinine <1.5 times ribosomal protein (Ser235/Ser236), rabbit polyclonal p-AKT (Ser473; Cell upper limit of normal and resolution of all related toxicities to grade V1 Signaling Technology, Boston, MA), and mouse monoclonal PTEN except for rash, which was required to improve to grade V 2. For (clone 6H2.1; Cascade Bioscience, Inc., Winchester, MA). The patients who develop DLT regardless of treatment cycle, the study EGFRpharmDx kit (DAKO Corp., Carpinteria, CA) was used for EGFR regimen was reduced to the dose level below that on which the patient was entered. Patients were removed from study for evidence of Primary antibodies were used at the following dilutions and progressive disease at any time after study initiation, grade 4 incubations: p-MAPK, 1:100 overnight at 4jC; p-S6 ribosomal protein, nonhematologic toxicity, more than two dose reductions due to 1:100 for 1 hour at room temperature; p-AKT, 1:50 overnight at 4jC; toxicity, dose reduction of gefitinib to <250 mg/d, noncompliance, or and PTEN, 1:1000 overnight at 4jC. The EGFR antibody was provided at a predetermined dilution, and immunohistochemistry was doneaccording to the Food and Drug Administration – approved manufac- turer’s protocol for the DAKO EGFRpharmDx kit.
Antiemetic therapy with ondansetron and dexamethasone was Immunohistochemistry. For all immunohistochemistry assays, 5-Am permitted if needed. Loperamide was prescribed for diarrhea as sections were cut from paraffin-embedded, formalin-fixed brain tissue, previously described (22). Hematopoietic growth factors and blood placed on silanized slides, deparaffinized with a series of xylenes, products were administered as indicated for hematologic DLT or cleared in a series of alcohols, and rehydrated. Endogenous peroxidase hematologic toxicity that occurred after cycle 1. Lipid lowering agents were permitted if prescribed before study enrollment, or for patients Antigen retrieval was done by one of several methods. For who developed either DLT or hyperlipidemia after cycle 1. Significant p-S6 and p-AKT, a solution of 10 mmol/L EDTA was used in a rash was treated with over-the-counter acne preparations, antihist- decloaking chamber for 5 minutes at 120jC. For p-MAPK and PTEN, a amines, and topical clindamycin and/or oral antibiotics (penicillins or sodium citrate solution (pH 6.0) was used in a Black and Decker Following antigen retrieval, slides were washed in TBS with 0.1% Tween 20, and nonspecific protein binding was blocked with 5% Venous blood samples (4 mL) were collected for gefitinib normal goat serum for 15 minutes at room temperature. For p-AKT pharmacokinetic studies from patients on days 7 and 10 of cycle and p-S6, a 30-minute incubation with goat anti-rabbit secondary 1 before the daily dose and 1, 2, 4, 6, 8, and 24 hours after the antibody was followed by detection with avidin-biotin complex Elite daily dose. For each sample, plasma supernatants were separated by kit (Vector Laboratories, Burlingame, CA). For PTEN, a 30-minute centrifugation (1,000 Â g for 10 minutes at room temperature) and incubation with goat anti-mouse supersensitive link was followed by immediately frozen at À20jC. Plasma concentrations of gefitinib detection with Super Sensitive Detection Kit (Biogenex, San Ramon, were determined by high-pressure liquid chromatography with CA). For MAPK, a 30-minute incubation with goat anti-rabbit tandem mass spectrometry detection by the Drug Metabolism and secondary antibody was followed by detection with the Multilink Pharmacokinetics Department at AstraZeneca, Alderley Park, United Detection kit (Biogenex, San Ramon, CA). Nuclear counterstaining was done using Harris’ modified hematoxylin. The intensity of Steady-state plasma drug concentrations were used to provide a cytoplasmic/membranous staining detected by immunohistochemistry measure of exposure and the pharmacokinetic variables. Maximum was scored on a scale of 0 to 4+, and the distribution was defined as steady-state plasma gefitinib concentration during the dosing interval the percentage of cells with any level of expression. Immunohisto- (Css,max), the time to reach maximum gefitinib concentration (Tmax), chemical staining was defined as ‘‘high’’ for tumors expressing 2 to 4+ and the minimum concentration during the dosing interval at steady intensity in z25% of tumor cells and as ‘‘low’’ for tumors expressing state (Css,min), defined as the concentration at 24 hours after dose on either 0 to 1+ staining in any percentage of tumor cells or 2 to 4+ each sample day, were obtained directly from the data. The area intensity in <25% of tumor cells (3).
under the concentration versus time curve at steady state (AUCss) was Dual-color FISH was done on formalin-fixed, paraffin- calculated by the linear trapezoidal rule using WinNonlin (Pharsight embedded tissue specimens using the EGFR/CEP 7, CEP 10/CEP 2 Clin Cancer Res 2006;12(3) February 1, 2006 Gefitinib Plus Sirolimus for Malignant Glioma (Vysis, Downers Grove, IL), and CEP 10/PTEN (Human BAC CITB Slides were viewed using an Olympus BX-60 fluorescent microscope.
library clone 265N13, Research Genetics, Huntsville, AL) probe The number of green and orange signals was enumerated in 100 intact, combinations (using three separate slides) for each patient sample.
nonoverlapping nuclei per slide. With regard to chromosomal gain, the CEP 2 was chosen as an internal control for the loss of chromosome 10 cutoff value was set at 20%, meaning that >20% of the enumerated (23). The EGFR probe does not discriminate between wild-type EGFR nuclei must show more than two copies of the respective probe. For chromosomal loss, the cutoff value was set at 30% for definitive loss Paraffin sections were cut at 5 Am onto silanized slides. Control and and 20% to 30% for indeterminate loss. EGFR gene amplification was patient slides were baked overnight at 56jC. Formalin-fixed, paraffin- defined as an EGFR/chromosome 7 centromere ratio of >2.0. Definitive embedded control cell lines, showing the locus of interest, were used as PTEN loss was defined as tumors in which z30% of nuclei exhibited less than two copies of the PTEN locus and two copies of CEP 2 Slides were deparaffinized, pretreated with 0.2 N HCl at room control. Indeterminate PTEN loss refers to tumors in which 20% to temperature for 20 minutes, then washed in deionized water and 2Â 30% of enumerated nuclei had less than two copies of the PTEN locus SSC for 3 minutes each. They were then placed in Pretreatment Solution (Vysis) at 80jC for 30 minutes and washed with two changesof 2Â SSC for 5 minutes each. Sections were subjected to digestion with protease at 37jC for 20 to 23 minutes. Slides were washed in twochanges of 2Â SSC for 5 minutes each and air-dried, then were denatured in a 70% formamide/2Â SSC solution at 72jC for 5 minutes malignant glioma were enrolled at the Duke University and immediately dehydrated in 70%, 85%, and 100% ethanol for 1 Medical Center between August 2004 and February 2005 minute each. Subsequently, the probe was denatured at 75jC for 5 (Table 2). Twenty-nine patients had glioblastoma multiforme minutes. Fluoresceinated probe was applied to each slide, sealed with (85%) and 5 (15%) had anaplastic astrocytoma. Fifteen rubber cement, and then placed in a humidified chamber at 37jC foran overnight incubation. After overnight incubation, slides were then patients (44%) were not on EIAEDs (stratum A) and 19 washed in 2Â SSC/0.3% NP40 at room temperature and then at 72jC (56%) were on EIAEDs (stratum B). Patient characteristics did for 2 minutes. 4V,6-Diamidino-2-phenylindole counterstain and a not differ substantially based on EIAED status. Twenty-three coverslip were applied to the hybridization area.
patients (68%) were male. The median age was 49.9 years Abbreviations: GBM, glioblastoma multiforme; GS, gliosarcoma; AA, anaplastic astrocytoma; KPS, Karnofsky performance status; XRT, X-ray therapy.
*EIAEDs: phenytoin, carbamazepine, phenobarbitol, oxcarbazepine, and primidone.
Clin Cancer Res 2006;12(3) February 1, 2006 (range, 32.8-76.8 years). All patients had a Karnofskyperformance status of at least 70%.
All patients had received prior X-ray therapy and chemo- therapy. The median number of prior chemotherapeutic agents administered per patient was 2 (range, 1-6). The median number of prior episodes of progressive disease per patient was 2 (range, 1-3). The median time from original diagnosis to initiation of study treatment was 29.8 weeks (range, 7.3-248.0 As of September 15, 2005, five patients continue to receive treatment on study with stable disease. Twenty-one patients and type of DLT observed at each dose level per stratum. Onegroup A patient developed fulminant progressive disease *Stratum A: patients not on EIAEDs (phenytoin, phenobarbitol, carbamaze- and discontinued study treatment after <2 weeks of cycle 1.
pine, oxcarbazepine, and primidone).
Although this patient did not experience a DLT, they were cOne additional patient was treated at the MTD (dose level1) for stratum A for replaced in the cohort for MTD determination. However, this bOne patient treated at dose level 2 of stratum A was not eligible for MTD patient was included in overall toxicity assessment. One determination due to fulminant progressive disease.
additional patient was added at dose level one for stratum A xOne patient treated at dose level 2 of stratum A and two patients treated at and provided additional safety and pharmacokinetic data.
dose level 3 of stratum B interrupted dosing during cycle 1and were replaced Three patients (one in dose level 2 of stratum A and two in dose level 3 of stratum B) decreased or interrupted dosing Stratum B: patients on EIAEDs (phenytoin, phenobarbitol, carbamazepine, during cycle 1 due to miscommunication or noncompliancewith administration guidelines. Although these patients wereassessable for DLT, they were not assessable for dose Limited sampling from two stratum B patients treated on escalation within each cohort and were therefore replaced.
dose level 3 was available and is therefore not included.
For stratum A, one of seven patients treated at dose level 1 Comparison of day 7 (gefitinib alone) and day 10 (gefitinib experienced DLT (grade 3 mucositis), whereas two of seven plus sirolimus) measures revealed that sirolimus, a known patients treated at dose level 2 experienced DLT, including one substrate for CYP3A4, did not affect gefitinib metabolism.
patient with grade 3 thrombocytopenia that required >2 weeks However, gefitinib exposure was significantly reduced by to resolve to retreatment criteria, and one patient with grade 3 concurrent EIAED use. Specifically, the dose-normalized, rash and mucositis. For stratum B, none of the patients geometric mean of AUCss for patients not on (stratum A) and experienced DLT at dose level 1. However, one of six patients for those on EIAEDs (stratum B) were 19.9 and 7.91 ng h/mL, treated at dose level 2 developed dose-limiting hypertriglycer- idemia, whereas two of eight patients treated at dose level 3 Trough sirolimus data was available on 23 patients, including developed DLT, including one patient with grade 3 diarrhea 12 patients from stratum A and 11 patients from stratum B. The and one patient with grade 3 mucositis.
mean trough sirolimus level for patients treated with 5 mg/d Non-DLT. One hundred courses of gefitinib plus sirolimus (7.0) was significantly less than that of patients treated with 10 have been administered to date, including 44 courses to mg/d (16.7; P < 0.0001); however, trough sirolimus levels did patients on stratum A and 56 courses to patients on stratum B.
not differ based on stratum (P = 0.136).
Table 4 summarizes the most frequent toxicities stratified by Archival tumor biomarker analysis. Archival tumor material toxicity grade and treatment stratum.
was available for 14 patients (Table 6). FISH analysis revealed Diarrhea, mucositis, and rash were the most common that 6 patients had EGFR amplification (43%) and 7 patients toxicities as expected. Hematologic toxicity and fasting had evidence of PTEN loss (50%). ‘‘High’’ levels of EGFR, cholesterol or triglyceride elevations were also noted, primar- p-S6, p-MAPK, and p-AKT were detected by immunohisto- ily as low-grade events, and also as infrequent grade 3 or 4 chemistry in 90% (9 of 10), 60% (6 of 10), 60% (6 of 10), events. Grade 1 or 2 infections, most frequently involving the and 90% (9 of 10) of assessable patients, respectively. A skin and nailbeds, were also noted. Two serious infections good correlation was observed between EGFR amplification occurred among patients on study and included episodes of detected by FISH and EGFR expression by immunohistochem- grade 3 and 4 pneumonia, respectively. The episode of grade 4 istry. All four tumors with EGFR amplification by FISH pneumonia was most likely due to aspiration following a showed 3 to 4+ EGFR expression in 90% to 100% of cells by seizure. Both events resolved with i.v. antibiotics and immunohistochemistry. Of note, three of four tumors with hospitalization. One patient, treated with gefitinib plus evidence of PTEN loss by FISH had elevated p-AKT expression sirolimus for 7 months, developed disseminated Aspergillus f2 months following study discontinuation while receiving Outcome. All 34 patients were evaluable for response. Two an alternative, salvage therapy. Of note, there were no grade 5 patients achieved a partial radiographic response, including one patient treated at dose level 1 on stratum A (Fig. 1) and Pharmacokinetic analyses. Ten patients from stratum A and another patient treated at dose level 3 on stratum B. Thirteen nine patients from stratum B underwent plasma gefitinib patients (38%) achieved stable disease, including 7 patients on stratum A (47%) and 6 patients on stratum B (32%). By Clin Cancer Res 2006;12(3) February 1, 2006 Gefitinib Plus Sirolimus for Malignant Glioma Table 4. Most frequent toxicities stratified by grade and patient stratum Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase.
*Stratum A: patients not on EIAEDs (phenytoin, carbamazepine, oxcarbazepine, phenobarbitol, and primidone).
cStratum B: patients on EIAEDs (phenytoin, carbamazepine, oxcarbazepine, phenobarbitol, and primidone).
bIncludes only grade 1and 2 events that occurred at a minimum of three times.
xIncludes all grade 3 and 4 events including those reported as DLT.
kCumulative number of events observed among all cycles of therapy.
{Percentage of all patients in stratum.
histology, 12 patients with glioblastoma multiforme (41%) weeks). Analysis of possible associations among clinical, and 1 with recurrent anaplastic glioma (20%) achieved stable pharmacokinetic, and archival tumor biomarker variables with outcome was limited by study accrual and the dose escalation With a median follow-up of 35.2 weeks (95% CI, 27.7-42.4), the median progression-free disease (PFS) and 6-month PFSrate for all patients were 8.2 weeks (95% CI, 7.5-18.6 weeks) and 23.5% (95% CI, 12.8-43.1%), respectively. Median PFSand 6-month PFS did not differ significantly by histology or The rationale for the study regimen of gefitinib plus sirolimus EIAED status. Among patients who achieved at least stable is that simultaneously targeting key upstream and downstream disease, the median PFS was 27.4 weeks (95% CI, 18.7-30.6 mediators of PI3K signaling may produce greater antitumor Table 5. Gefitinib pharmacokinetic variables Abbreviations: Gmean, geometricmean; CV%, coefficient of variation.
*EIAEADs: phenytoin, carbamazepine, oxcarbazepine, phenobarbitol, primidone.
Clin Cancer Res 2006;12(3) February 1, 2006 activity than that achieved when either mediator is targeted downstream signaling mediators or alternative signaling separately. Results of preclinical studies confirm that such pathways may provide compensatory proliferative and survival combinatorial regimens are capable of synergistic antitumor activity (9, 24 – 27). Furthermore, such combinatorial regi- Our phase I study achieved its primary objective of mens may be less vulnerable to resistance mechanisms against establishing the MTD of a continuous daily dosing regimen targeted therapeutics. Although current understanding of such of gefitinib plus sirolimus for patients with recurrent malignant resistance mechanisms is limited (28), insights can be gained glioma. Specifically, the MTD is 500 mg of gefitinib plus 5 mg from both clinical and preclinical studies. For example, the of sirolimus for patients not on EIAEDs, and 1,000 mg of majority of patients exhibit resistance to EGFR TKIs, although gefitinib plus 10 mg of sirolimus for those on EIAEDs.
most glioblastoma multiforme tumors express EGFR, suggest- Furthermore, we show that these agents can be safely ing that compensatory mechanisms can overcome EGFR combined at doses used in monotherapy dosing schedules inhibition. One such compensatory mechanism may be (6, 41, 42). There were no unexpected toxicities and the increased activity of additional growth factor receptors.
spectrum of observed toxicities, including DLTs, was similar to Growth factors reported to be overexpressed in malignant those previously reported in monotherapy studies (6, 12, 13, glioma include platelet-derived growth factor receptor (29, 41, 42). Although not observed among enrolled patients, 30), vascular endothelial growth factor (31), fibroblast growth opportunistic infections pose an appropriate concern with this factor receptor (32), and insulin-like growth factor-I receptor regimen due to the immunosuppressive activity of sirolimus, (33). Furthermore, some EGFR-resistant tumors exhibit particularly because malignant glioma patients are inherently activation of alternative growth factor receptor pathways, immunocompromised (43, 44) and are frequently on immu- suggesting that either tumor mitogenesis or induction of angiogenesis may act to compensate for EGFR signaling loss Secondary objectives of this study included the evaluation of (26, 33 – 36). EGFR TKI resistance has also been associated pharmacokinetic end points, the assessment of biomarkers with increased activity of intracellular mediators. For example, from archival tumor specimens of enrolled patients, and the loss of the PTEN tumor suppressor, which constitutively determination of evidence of antitumor activity. Our phar- activates AKT (36, 37), is linked to resistance to EGFR-based macokinetic studies confirmed that gefitinib exposure is therapies (25, 38 – 40). In a recent glioblastoma multiforme significantly affected by concurrent EIAED use and provided trial, elevated levels of p-AKT correlated with erlotinib reassurance that sirolimus does not affect gefitinib metabolism.
resistance (7). These data suggest that in response to upstream The analysis of our immunohistochemistry and FISH growth factor TKI therapy, enhanced activity of either findings was limited by specimen availability and the dose Table 6. Archival tumor biomarker analysis Abbreviations: ND, not done; PR: partial response; SD: stable disease; PD: progressive disease; TTP: time to progression; I, intensity; D, distribution.
*FISH: amplified EGFR : EGFR/chromosome 7 ratio >2.0; polysomy EGFR : EGFR/chromosome 7 ratio 1-2; PTEN loss: <2 copies PTEN with 2 copies of CEP 2 control in z30% nuclei; PTEN indeterminate: <2 copies PTEN with 2 c opies of CEP 2 c ontrol in 20% to 30% nuc lei.
cImmunohistochemistry: wild-type EGFR, p-S6 ribosomal protein, p-p44/42 MAPK, p-AKT. I, most common staining pattern present overall (0+, no staining; 1+, minimal cytoplasmic/membraneous staining; 2+, mild cytoplasmic/membraneous staining; 3+, moderate cytoplasmic/membraneous staining; 4+, strong cytoplasmic/membraneous staining). D, percent positive cells; focal refers to heterogeneous, regional, or sporadic staining of <25% of evaluated tumor cells.
bStratum A: no EIAEDs; stratum B, on EIAEDs.
Clin Cancer Res 2006;12(3) February 1, 2006 Gefitinib Plus Sirolimus for Malignant Glioma dynamic measures to assess the study regimen’s effect onintratumoral PI3K and mTOR signaling was not assessed.
Therefore, confirmation that either study agent was success-fully delivered at dose levels required to inhibit the intendedintracellular target was not obtained. Ongoing and plannedclinical trials with EGFR and mTOR inhibitors that incorpo-rate pharmacodynamic evaluations of tumor cell targets mayclarify this critical issue. Finally, it is possible that suppress-ing both EGFR and mTOR may not be sufficient to effectivelytreat some glioblastoma multiforme tumors due to aberrantactivation of alternative downstream PI3K mediators or othergrowth factor/survival pathways. The identification of severalsignal transduction pathways commonly altered in malignantglioma suggests that targeting pathways in parallel may alsocontribute to effective therapeutic synergy.
In conclusion, we report the first clinical trial incorporating a combinatorial regimen of signal transduction inhibitors formalignant glioma patients. In addition to establishing theMTD of this regimen, we confirm that therapeutics targetingEGFR and mTOR can be safely coadministered to malignantglioma patients. Phase 2 trials to evaluate the antitumoractivity of EGFR and mTOR targeting regimens are under wayfor recurrent malignant glioma patients. Combinatorialregimens, including those designed to simultaneously target Fig.1. Partial response to gefitinib plus sirolimus. Top and middle, axial T1-weighed key upstream and downstream signaling mediators, represent sequences following gadolinium administration; bottom,T2-weighed sequences.
After one cycle of treatment, a marked reduction of both contrast-enhancing tumor an important advance in the evaluation of targeted therapeu- and associated edema was observed. The radiographic response, accompanied by tics for cancer patients. The therapeutic potential of such marked clinical improvement, was maintained for 4 months, at which point aprogressive tumor developed.
combinatorial approaches for future studies is noteworthy butcritically hinges on the comprehensive integration of clinical,pretreatment tumor biomarker, pharmacokinetic and intra- escalation design of this study. Furthermore, tumor samples evaluated in our trial were obtained at either initial diagnosisor after prior therapy and therefore may not have reflected theactual molecular genetic profile of the tumor at study entry.
Nonetheless, the potential of such assays to prospectively identify appropriate cohorts of malignant glioma patientsfor treatment with selected targeted therapeutics was recently shown (7). In this analysis, patients with archival tumorsamples showing p-AKT and EGFR amplification had a significantly greater likelihood of response to the EGFR TKI The rate of radiographic response on the current study was comparable with that observed among glioblastoma multi- forme patients treated with temozolomide at first recurrence (45). However, PFS on the current study was similar to that achieved on our prior phase II study with gefitinib alone (6).
Although the assessment of antitumor activity is limited in any phase I study, several additional factors may have affected our study’s outcome. First, patients were heavily pretreated, having enrolled following treatment with a median of two prior chemotherapy agents (range, 1-6) and a median of two prior recurrences (range, 1-3). Second, nearly all patients on the current study had bulky measurable tumor, whereas only 11 of 53 patients (21%) enrolled onour prior phase 2 study had measurable tumor (6). Third, Abbreviations: PR, partial response; SD, stable disease; PD, progressive EGFRvIII expression, which was unable to be assessed in the current study due to technical factors with the EGFRvIII *% Patients with an evaluable assay.
immunohistochemistry assay, may have also affected re- cAmplified: EGFR/chromosome 7 centromere ratio of >2.0.
sponse (7, 46). Fourth, our pharmacokinetic studies confirm bLoss: 30% of nuclei with definitive loss and 20-30% with indeterminate loss.
x High, 1-4+ intensity in z25% of tumor cells; low, either no staining or 1-4+ that concurrent use of EIAEDs markedly diminish gefitinib intensity in <25% of tumor cells.
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