Phase 2 trial of erlotinib plus sirolimus in adults with recurrent glioblastoma

Abstract

We evaluated the anti-tumor activity and safety of erlotinib, a receptor tyrosine kinase inhibitor of the epidermal growth factor receptor, plus sirolimus, an inhibitor of the mammalian target of rapamycin, among patients with recurrent glioblastoma (GBM) in a phase 2, open-label, single-arm trial. Thirty-two patients received daily erlotinib and sirolimus. The doses of erlotinib and sirolimus were 150 mg and 5 mg for patients not on concurrent CYP3A-inducing anti-epileptics (EIAEDS), and 450 mg and 10 mg for patients on EIAEDS. Evaluations were performed every two months. The primary endpoint was 6-month progression-free survival and secondary endpoints included safety and overall survival. Archival tumor samples were assessed for EGFR, EGFRvIII, PTEN, pAKT and pS6. Enrolled patients were heavily pre-treated including 53% who had received three or more prior chemotherapy agents and 28% who had received prior bevacizumab therapy. The most common grade > or = 2 adverse events were rash (59%), mucositis (34%) and diarrhea (31%). Grade 3 or higher events were rare. Best radiographic response included stable disease in 15 patients (47%); no patients achieved either a CR or PR. The estimated 6-month progression-free survival was 3.1% for all patients. Progression-free survival was better for patients not on EIAEDs (P = 0.03). Tumor markers failed to show an association with PFS except for increased pAKT expression which achieved borderline significance (P = 0.045). Although neither rash nor diarrhea had an association with outcome, hyperlipidemia was associated with longer PFS (P = 0.029). Erlotinib plus sirolimus was well tolerated but had negligible activity among unselected recurrent GBM patients. (ClinicalTrials.gov number: NCT0062243).

Full text

Phase 2 trial of erlotinib plus sirolimus in adults with recurrent
glioblastoma
David A. Reardon,
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Department of
Pediatrics, Duke University Medical Center, Durham, NC 27710, USA
Annick Desjardins,
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
James J. Vredenburgh,
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
Sridharan Gururangan,
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Department of
Pediatrics, Duke University Medical Center, Durham, NC 27710, USA
Allan H. Friedman,
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
James E. Herndon II,
Cancer Center Biostatistics, Duke University Medical Center, Durham, NC 27710, USA
Jennifer Marcello,
Cancer Center Biostatistics, Duke University Medical Center, Durham, NC 27710, USA
Julie A. Norfleet,
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
Roger E. McLendon,
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA
John H. Sampson, and
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
Henry S. Friedman
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Department of
Pediatrics, Duke University Medical Center, Durham, NC 27710, USA
Abstract
We evaluated the anti-tumor activity and safety of erlotinib, a receptor tyrosine kinase inhibitor of
the epidermal growth factor receptor, plus sirolimus, an inhibitor of the mammalian target of
rapamycin, among patients with recurrent glioblastoma (GBM) in a phase 2, open-label, single-arm
trial. Thirty-two patients received daily erlotinib and sirolimus. The doses of erlotinib and sirolimus
were 150 mg and 5 mg for patients not on concurrent CYP3A-inducing anti-epileptics (EIAEDS),
and 450 mg and 10 mg for patients on EIAEDS. Evaluations were performed every two months. The
primary endpoint was 6-month progression-free survival and secondary endpoints included safety
and overall survival. Archival tumor samples were assessed for EGFR, EGFRvIII, PTEN, pAKT and
D. A. Reardon, The Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Box 3624, Durham, NC 27710,
USA reard003@mc.duke.edu.
NIH Public Access
Author Manuscript
J Neurooncol. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
J Neurooncol. 2010 January ; 96(2): 219–230. doi:10.1007/s11060-009-9950-0.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

pS6. Enrolled patients were heavily pre-treated including 53% who had received three or more prior
chemotherapy agents and 28% who had received prior bevacizumab therapy. The most common
grade ≥2 adverse events were rash (59%), mucositis (34%) and diarrhea (31%). Grade 3 or higher
events were rare. Best radiographic response included stable disease in 15 patients (47%); no patients
achieved either a CR or PR. The estimated 6-month progression-free survival was 3.1% for all
patients. Progression-free survival was better for patients not on EIAEDs (P = 0.03). Tumor markers
failed to show an association with PFS except for increased pAKT expression which achieved
borderline significance (P = 0.045). Although neither rash nor diarrhea had an association with
outcome, hyperlipidemia was associated with longer PFS (P = 0.029). Erlotinib plus sirolimus was
well tolerated but had negligible activity among unselected recurrent GBM patients.
Keywords
Malignant glioma; EGFR; mTOR; Erlotinib; Sirolimus
Introduction
The current standard of care for newly diagnosed glioblastoma (GBM) patients, maximal safe
resection followed by radiation therapy and temozolomide, results in a median overall survival
(OS) of only 14.6 months, and a median progression free survival (PFS) under 7 months [1].
Although bevacizumab with or without irinotecan chemotherapy has recently been associated
with modest survival benefit for recurrent patients [2–4], death following progression is
essentially universal as salvage therapies are ineffective [5–7]. Therefore, there remains a
significant unmet need for innovative, more effective treatments to improve outcome for
recurrent GBM patients.
Activation of the phosphatidylinositol 3′-kinase (PI3K) signal transduction pathway enhances
tumor cell proliferation, migration, angiogenesis and survival. Dysregulated PI3K signaling
occurs commonly in GBM [8,9] due to activation of cell surface, growth factor receptors such
as the epidermal growth factor receptor (EGFR), or functional loss of the phosphatase and
tensin homolog (PTEN) tumor suppressor gene [10], and confers a poor prognosis following
standard cytotoxic therapy [11]. The mammalian target of rapamycin (mTOR) is a key
downstream mediator of the PI3K pathway, and provides critical regulation of several essential
cellular processes in both normal and neoplastic cells including nutrient metabolism, cell-cycle
progression and protein translation [12].
Erlotinib (Tarceva; Genentech, Inc., South San Francisco and OSI Pharmaceuticals, Melville,
NY), an orally active, reversible EGFR tyrosine kinase inhibitor, is currently approved by the
USA Food and Drug Administration for recurrent non-small cell lung cancer [13,14] and in
combination with gemcitabine for pancreatic cancer [15]. Sirolimus (Rapamycin; Rapamune®,
Wyeth Pharmaceuticals, Ayerst, PA), first isolated from the soil bacteria Streptomyces
hygoscopicus, was approved by the FDA in 1999 as an immunosuppressant to prevent rejection
of solid organ transplants. Sirolimus binds to the FK binding protein complex (FKBP-12)
thereby preventing mTOR activation. Sirolimus and its analogues can induce either G1 phase
arrest or apoptosis in many human tumor cell lines including GBM [16–22] and have anti-
tumor activity against malignant glioma and medulloblastoma xenografts [23,24].
Although EGFR and mTOR are key PI3K signaling mediators, the anti-tumor benefit of single-
agent antagonists against either EGFR or mTOR among unselected, recurrent GBM patients
has been disappointing [25–32]. Rationally designed combinatorial regimens represent a
potential strategy to improve efficacy of such compounds. Our group and others have
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demonstrated that combination of an EGFR antagonist with an mTOR inhibitor leads to
synergistic anti-tumor activity in GBM xenografts [33,34].
We conducted the current phase 2 study to determine the anti-tumor benefit of erlotinib
combined with sirolimus as a strategy to simultaneously inhibit key upstream and downstream
mediators of the PI3K signaling pathway among recurrent GBM patients.
Materials and methods
Protocol objectives
The primary objective was to define the anti-tumor activity as measured by progression-free
survival at 6 months (PFS-6) of erlotinib plus sirolimus in adults with recurrent GBM.
Secondary objectives included the assessment of additional efficacy endpoints including
radiographic response rate, median PFS and median OS following treatment with this regimen,
and to further define the toxicity of this combinatorial approach in recurrent GBM patients.
Patient eligibility criteria
Patients were required to have histologically confirmed GBM that was radiographically
progressive following prior radiation or chemotherapy. Additional enrollment criteria
included: age at least 18 years; KPS ≥ 70%; stable corticosteroid dose for at least 1 week prior
to therapy initiation; hematocrit >29%; absolute neutrophil count >1,500 cells/μl; platelet count
>100,000 cells/μl; and serum creatinine, aspartate aminotransferase and bilirubin <1.5 times
the institutional upper limit of normal. Patients were also required to be at least 2 weeks from
prior surgical resection, 12 weeks from prior radiotherapy (unless histopathologic confirmation
of recurrent GBM was obtained or new enhancement outside the radiation field was
demonstrated on MRI) and 4 weeks from prior chemotherapy (6 weeks for nitrosoureas).
Patients treated with prior anti-angiogenic therapy were eligible, however a minimum of 6
weeks was required from prior bevacizumab dosing. All patients were also required to have
recovered from toxicities of prior therapy and to provide informed consent.
Patients were excluded for any of the following: prior therapy with either an EGFR or mTOR
antagonist; uncontrolled intercurrent illness including active infection requiring intravenous
antibiotics, symptomatic congestive heart failure, unstable angina, grade 3 or greater
hyperlipidemia or significant gastrointestinal, renal or liver disease; pregnancy or nursing; and
refusal to use effective contraception if of reproductive potential.
Treatment design
Erlotinib and sirolimus were orally administered on a continuous daily dosing schedule of 28
day cycles. Erlotinib and sirolimus metabolism are enhanced by concurrent use of CYP3A-
inducing anti-epileptic drugs (EIAEDs) including phenytoin, carbamazepine, phenobarbital,
oxcarbazapine and primidone [26,35–37]. Erlotinib was dosed at the MTD established
previously for patients based on concurrent EIAED use [26] while the sirolimus dose was based
on dosing that was safely administered with an EGFR inhibitor in prior studies [37] and that
was associated with mTOR inhibition in patient tumor samples [28]. The daily erlotinib and
sirolimus doses were 150 mg and 5 mg for patients not on EIAEDs, while those on EIAEDs
took 450 mg and 10 mg/day, respectively. A study therapy continued for up to 12 cycles unless
patients developed unacceptable toxicity, tumor progression, non-compliance with protocol
guidelines or consent withdrawal.
Supportive care
Antiemetic therapy with ondansetron and dexamethasone was permitted. Loperamide was
prescribed for diarrhea as previously described [38]. Hematopoietic growth factors and blood
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products were administered as indicated for hematologic toxicity. Lipid lowering agents were
permitted for documented hyperlipidemia. Significant rash was treated with over the counter
acne preparations, antihistamines as well as topical clindamycin and/or oral antibiotics
(penicillins or cephalosporins) as needed.
Study assessments
Study investigators determined response by neurologic examination and contrast-enhanced
MRI prior to the start of every other treatment cycle according to the Macdonald criteria [39].
A complete response (CR) was defined as disappearance of all enhancing tumor on consecutive
MRIs at least 6 weeks apart, with corticosteroid discontinuation and neurologic stability or
improvement. A partial response (PR) was defined as ≥50% reduction in size (product of largest
perpendicular diameters) of enhancing tumor on consecutive MRIs at least 6 weeks apart, with
stability or improvement of neurologic status and corticosteroid requirement. Progressive
disease (PD) was defined as ≥25% increase of enhancing tumor, a new enhancing lesion or
significant clinical decline. Stable disease (SD) was defined as any assessment not meeting
CR, PR, or PD criteria.
Toxicities were graded according to the National Cancer Institute's Common Toxicity Criteria
Version 3.0 and classified as related to the study regimen unless attributable to either underlying
tumor progression, concurrent medical condition or concomitant medication.
Dose modification and retreatment criteria
The daily erlotinib dose was reduced by 50 mg for patients not on EIAEDs, and 100 mg for
those on EIAEDs who developed intolerable grade 3 rash, grade 3 diarrhea or other grade ≥3
attributable toxicity. Erlotinib was discontinued for grade 4 rash or diarrhea. The daily
sirolimus dose was reduced by 50% for grade 4 neutropenia, grade 3 thrombocytopenia, grade
3 hyperlipidemia or other grade ≥3 attributable toxicity. If the event recurred, the sirolimus
dose was further reduced to 3 times per week (Monday–Wednesday–Friday).
Initiation of each cycle required an ANC ≥1,000/mm3; a platelet count ≥ 1,000/mm3; AST,
bilirubin and creatinine <twice the institutional upper limit of normal; resolution of any related
grade ≥3 event to grade ≤1 except for rash or diarrhea which were required to resolve to grade
≤2.
Archival tumor biomarker assessment
Archival tumor samples from either initial diagnosis or after prior therapy, were analyzed for
phosphorylated mitogen-activated protein kinase (p-MAPK), phosphorylated-S6 ribosomal
protein (p-S6), phosphorylated-AKT (p-AKT), PTEN, EGFR and the EGFRvIII mutant
receptor using immunohistochemistry (IHC) reagents and methods as previously described
[37]. Similarly archival tumor samples were analyzed by fluorescence in situ hybridization
(FISH) for EGFR and PTEN DNA locus copy number using reagents and methods as
previously described [37]. Immunohistochemistry assays were scored for intensity of
cytoplasmic/membranous staining detected by IHC was scored on a scale of 0–4+ and the
distribution was defined as the percentage of cells with any level of expression. IHC staining
was defined as “high” for tumors expressing 2–4+ intensity in 25% or more of tumor cells and
as “low” for tumors expressing either 0–1+ staining in any percentage of tumor cells or 2–4+
intensity in less than 25% of tumor cells [8].
For FISH studies, the cutoff value for chromosomal gain was set at 20%, meaning that greater
than 20% of the enumerated nuclei must show greater than two copies of the respective probe.
For chromosomal loss, the cutoff value was set at 30% for definitive loss and 20–30% for
indeterminate loss. EGFR gene amplification was defined as an EGFR/chromosome 7
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centromere ratio of greater than 4.0. Definitive PTEN loss was defined as tumors in which
≥30% of nuclei exhibited <2 copies of PTEN locus and 2 copies of CEP 2 control. Indeterminate
PTEN loss refers to tumors in which 20-30% of enumerated nuclei had <2 copies of PTEN
locus and 2 copies of CEP 2 control.
Statistical considerations
The primary goal of this study was to evaluate the 6-month PFS rate of erlotinib plus sirolimus
in the treatment of patients with recurrent or progressive GBM. Yung reported a median 6-
month PFS of 21% (95% CI: 13.29) among recurrent GBM patients undergoing treatment with
temozolomide at first recurrence [36]. The sample size goal of 32 patients for the current study
provided 90% power with a test conducted at the 0.10 level of significance to differentiate
between a 6-month PFS of 5 and 20%.
“Stopping rules” for unexpectedly poor efficacy and unacceptable toxicity were incorporated
for each stratum. Specifically, if 10 or more of the first 16 patients per stratum experienced
progression or death within 2 months of study initiation further accrual would be suspended.
In addition, if six or more of the first 16 patients per stratum experienced unacceptable toxicity,
defined as grade ≥4 non-hematologic events, further accrual would be suspended.
Progression-free survival (FFP) and overall survival (OS), measured from the date of cycle 1
treatment administration, were summarized using the Kaplan–Meier estimator including 95%
CIs. A log-rank test was used to assess the relationship of adverse events (grade ≥ 2 rash,
diarrhea, or hyperlipidemia) and tumor markers (EGFR, PTEN, pAKT, pS6 and pMAPK) on
PFS.
Results
Patient characteristics
Thirty-two patients with recurrent GBM were enrolled at Duke University Medical Center
between May 2007 and March 2008 (Table 1). Twenty-four patients (75%) were not on
EIAEDs (stratum A) and 8 (25%) were on EIAEDs (stratum B). Patient characteristics did not
differ substantially based on EIAED status. The median age was 54 years (range, 40–71 years).
All patients had a KPS of at least 70%.
All patients had received prior XRT and chemotherapy. Nineteen patients (59%) had more
than one prior episode of progressive disease and 17 patients (53%) had received three or more
prior chemotherapy agents. Eleven patients received prior anti-angiogenic therapy including
9 (28%) patients treated with bevacizumab and two patients treated with sorafenib. The median
time from original diagnosis to initiation of study treatment was 54.4 weeks (range, 19.6–158
weeks).
As of 4/1/09, all patients have discontinued study therapy. Thirty patients have died due to
progressive tumor.
Toxicity
Sixty-four courses of erlotinib plus sirolimus were administered to study patients including 54
courses to patients on stratum A and 10 courses to patients on stratum B. The most frequent
grade ≥2 toxicities were rash (59%), mucositis (34%), diarrhea (31%), fatigue (28%) and
hyperlipidemia (25%) (Table 2). Most toxicities were grade 2. Only one grade 4 event
occurred––reversible thrombocytopenia in a patient who had received seven prior
chemotherapeutic agents. There were no grade 5 events. Hematologic and electrolyte
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abnormalities were occasionally noted. Four patients developed infections; three of these were
grade 2, while the remainder was a grade 3 urinary tract infection.
Archival tumor biomarker analysis
Archival tumor material was available for 22 patients (69%). Analysis by FISH revealed EGFR
amplification in four of nine tumors (31%), whereas 11 of 22 tumors (50%) had evidence of
PTEN loss. Analysis by IHC revealed that all evaluated tumors had “high” EGFR expression,
including 7 of 15 tumors with 2–3+ staining in >90% of cells and 8 of 15 tumors with 2–3
staining in >25% of cells. Interpretation of IHC was consistent with PTEN loss in 12 of 20
(60%) evaluable tumors. Expression of EG-FRvIII, pAKT, pS6 and pMAPK was detected in
25%, 44%, 83% and 40% of assessed tumors, respectively (Fig. 1).
Among tumors with PTEN loss by FISH that were also assessable for pAKT and pS6, five of
eight (63%) had detectable pAKT and six of eight (75%) had detectable pS6. Among tumors
with EGFRvIII expression that were also assessable for pAKT and pS6, only one of four (25%)
had detectable pAKT while three of three had detectable pS6.
Outcome
Outcome is summarized in Table 3. All 32 patients were evaluable for response. No patients
achieved a radiographic response however stable disease was achieved by 13 patients on
stratum A (54%) and two patients on stratum B (25%).
With a median follow-up of 69.3 weeks (95% CI, 62-87.4), the median PFS and 6-month PFS
rate for all patients were 6.9 weeks (95% CI, 3.9-11.0 weeks) and 3.1% (95% CI, 0.2–13.7%),
respectively (Fig. 2a). Median PFS and 6-month PFS for patients on stratum A were 8.4 weeks
(95% CI, 3.9–12.1 weeks) and 4.2% (95% CI, 0.3–17.6%), respectively, and 4.0 weeks (95%
CI, 3.9–7.4 weeks) and 0%, respectively for patients on stratum B. A comparison of stratum-
specfic PFS was statistically significant (P = 0.03).
Median PFS for patients who received prior bevacizumab (n = 7) was 4.0 weeks (95% CI: 3.6,
7.0), while the median PFS for those who did not receive prior bevacizumab (n = 25) was 7.4
weeks (95% CI: 3.9, 11.9) (P = 0.18). None of the archival tumor sample markers showed an
association with 6-month PFS except for expression of p-AKT, which achieved borderline
significance (p = 0.045). However, tumor biomarker analysis was clearly limited by the small
number of analyzed samples and the overall low activity of the study regimen. Among
treatment-specific toxicities, PFS was associated with hyperlipidemia (P = 0.03) but not rash
or diarrhea.
Median OS for all patients was 33.8 weeks (95% CI, 21.9-53.6 weeks) and did not differ
significantly by EIAED status (Fig. 2b).
Discussion
Nearly all newly diagnosed GBM patients progress within months of diagnosis despite
aggressive, multi-modality therapy. Historically, salvage therapies have been largely
ineffective with most patients dying within months of recurrence. Many groups, including ours,
have extensively evaluated salvage regimens incorporating recently developed therapeutics
designed to inhibit key mediators of cell signaling pathways, including the PI3-Akt and ras-
MAPK transduction pathways. These pathways are dysregulated in the majority of GBM tumor
samples and are known to critically contribute to GBM pathophysiology including cell
survival, proliferation, invasion, and angiogenesis. Furthermore, evidence of activation of these
signaling pathways confers a poorer outcome [11,40].
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Unfortunately, clinical trials evaluating inhibitors of signal transduction pathways,
administered as single-agents among unselected, recurrent GBM patients, have been largely
disappointing [25–27,29,30,41–43]. Several factors may underlie the poor outcome of these
trials including heterogeneity of target expression within and across tumors, complexity of
signaling cascades including redundancy and cross-talk, and resistance mediated by
compensatory upregulation of alternative pathway mediators.
The current study was designed to evaluate erlotinib, an EGFR tyrosine kinase inhibitor, when
combined with sirolimus, an mTOR inhibitor among recurrent GBM patients. The hypothesis
underlying this combinatorial regimen is that simultaneously targeting upstream and
downstream mediators is more likely to suppress PI3 K-AKT signaling and thereby induce
greater tumor cell death than either agent alone. Furthermore, this combination may better
overcome target heterogeneity, mitigate the impact of signaling pathway redundancy and cross-
talk, and blunt resistance mediated by upregulation of alternative pathway mediators.
Results of preclinical studies demonstrate that combinatorial regimens can simultaneously
inhibit dual targets within the PI3K-A pathway and provide enhanced anti-tumor efficacy.
Combination of an EGFR inhibitor with inhibitors of either AKT or PI3K led to diminished
cell proliferation [44,45]. Similarly enhanced anti-glioma efficacy was observed with dual PI3
kinase/mTOR targeting [46]. We previously demonstrated that combination therapy with
AEE788 (Novartis Pharma AG, Basel, Switzerland), an EGFR TKI, with the rapamycin-
derivative, RAD001 (Novartis Pharma AG, Basel, Switzerland) increased cell cycle arrest and
apoptosis with reduced proliferation in vitro compared to either agent against GBM cell lines.
Furthermore, the combination improved tumor growth inhibition and overall survival in
athymic mice with GBM xenografts [34]. Synergistic inhibition of glioma cell growth was also
demonstrated following administration of sirolimus with the EGFR TKI EKI-785 (Wyeth
Pharmaceuticals, Madision, New Jersey, USA) [33]. Finally, sirolimus and erlotinib achieved
enhanced tumor cell death and growth inhibition, as well as blocking downstream PI3K
pathway signaling in both PTEN-deficient and PTEN-intact GBM cells [47].
Clinical studies evaluating simultaneous administration of EGFR and mTOR inhibitors among
recurrent GBM patients are limited. Doherty reported a retrospective review of 28 recurrent
malignant glioma patients (GBM, n = 22; grade 3 anaplastic glioma, n = 6) treated with
sirolimus plus either gefitinib or erlotinib. Of note, none of the patients were on EIAEDs. Partial
responses were observed in five patients (19%) including four with GBM (18%), and stable
disease was achieved in 14 patients (50%) including nine with GBM (41%). PFS-6 for the
GBM patients was 25% [48]. Similarly, Kreisl noted partial radiographic response and stable
disease in 3 (14%) and 8 (36%) of recurrent GBM patients treated with everolimus and
gefitinib, however durability of disease control was poor with a median PFS of only 2.6 months
and only one patient (4%) remaining progression-free at six months [49]. We recently
demonstrated that the EGFR tyrosine kinase inhibitor gefitinib can be safely co-administered
at standard single-agent dose levels in combination with sirolimus among recurrent malignant
glioma patients. In that study, we also confirmed that gefitinib pharmacokinetics are not
affected by sirolimus co-administration. Furthermore, despite the phase I design of this study,
modest evidence of anti-tumor activity was observed including a PFS-6 of 23.5% [37].
Unfortunately, enthusiasm for further clinical development of gefitinib dampened upon
revocation of accelerated approval status by the FDA following failure to demonstrate adequate
anti-tumor activity in randomized phase 3 studies for recurrent non-small cell lung cancer
[50,51].
Despite the encouraging preclinical and clinical data supporting the rationale of combining
erlotinib with sirolimus for recurrent GBM, results of the current study were disappointing.
Several factors may have contributed. First, patients in the current study were heavily
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pretreated. Fifty-nine percent of patients enrolled after two or more prior episodes of
progressive disease and 75% had received two or more prior chemotherapeutics, while 28%
had failed prior bevacizumab therapy. Second, the dose of erlotinib used in this phase 2 study
was based on the MTD of single-agent erlotinib [26], while we used a sirolimus dose that has
been safely administered in prior studies [37] and associated with mTOR inhibition in patient
tumor samples [28]. A phase I study of this regimen in this patient population has not been
performed; thus the true MTD has not been defined. In addition, an optimal biologic dose has
also not been determined. One strategy to determine an optimal biologic dose involves
demonstration of target inhibition in GBM cells obtained from patients treated prior to planned
debulking surgery. Of note, an analysis of tumor samples obtained from a sampling of recurrent
GBM patients failed to identify an effect of pre-treatment with either gefitinib or erlotinib on
EGFR activity in vivo [52]. These findings suggest that evaluation of alternative intra-tumoral
pharmacodynamic molecular effects may be more informative for patients treated with a
combination of EGFR and mTOR inhibitors. The critical importance of intratumoral
pharmacodynamic assessment is underscored by the recent report of increased AKT activation
observed among a subset of patients following mTOR inhibitor therapy [28]. Increased Akt
activation and increased tumor cell survival pathway signaling is associated with mTOR
inhibitor therapy [53,54] and may contribute to failure of mTOR inhibitor therapy in some
cancer patients.
Finally, pharmacokinetic analyses were not incorporated into this study. It is possible that since
both erlotinib and sirolimus are CYP 3A substrates [35,36], a detrimental pharmacokinetic
interaction between these two agents may have occurred which led to limited anti-tumor
efficacy. This interaction may have been further compounded by EIAEDs which were
administered to 8 (25%) patients on this study. In fact, we noted that PFS was modestly
improved among patients not on EIAEDs (P = 0.03). In addition, intra-tumoral
pharmacokinetics are particularly relevant in malignant glioma patients because the blood brain
barrier (BBB) may limit CNS penetration even when adequate systemic levels are achieved.
In particular, it appears that several TKIs, including erlotinib, are substrates for BBB efflux
proteins such as p-glycoprotein (P-gp), breast cancer resistance protein (BCRP, ABCG2) and
multidrug resistance protein 2 (MRP2; ABCC2) [55,56].
Although some studies have noted a correlation between tumor marker expression and outcome
[32,57–59], others studies have failed to detect such an association [25,52,60,61]. In the current
study, we did not observe a relationship between PI3/Akt pathway marker expression and
outcome with the exception of increased pAKT, which achieved borderline significance.
Paradoxically, others have noted an association between low pAKT and response to EGFR
inhibitor therapy [58,59]. An explanation for these discrepant findings is unclear, but may
reflect differences in either immunohistochemistry assay methods, thresholds used to define
expression levels or response criteria. Similarly, although increased sensitivity to mTOR
inhibition has been reported among PTEN-deficient glioma lines compared to those with intact
PTEN [62], others have reported that response to mTOR inhibition does not correlate with
PTEN status [63], and a recently completed clinical trial of single-agent CCI-799
(temsirolimus) found no correlation between PTEN status and outcome [27]. An important
factor limiting analyses that attempt to associate tumor biomarkers and outcome, including that
performed in the current study, is the use of archival tumor material rather than tumor material
obtained immediately prior to study treatment. While the latter accurately reflects the
constellation of molecular genetic abnormalities that characterize a given tumor at the time of
treatment, archival tumor material may do so less reliably.
Although some studies have noted an associaton between either rash [32] or diarrhea [25] and
outcome following EGFR inhibitor therapy, our study was consistent with others that did not
observe an association between outcome and either rash or diarrhea [61,64]. However, we did
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observe a correlation between hyperlipidemia and improved outcome as was similarly noted
among recurrent GBM patients treated with the mTOR inhibitor temsirolimus [27]. Although
it is possible that this association occurred by chance due to multiple hypothesis testing, future
studies should further evaluate the association between hyperlipidemia and response to mTOR
inhibitor therapy.
Overall, the combination of erlotinib and sirolimus was well tolerated among patients treated
on the current study. The range of toxicities was within that predicted to be observed with
single-agent administration of each of these agents [26,28], as well as within the spectrum of
what we observed previously among recurrent GBM patients treated with gefitinib and
sirolimus [37]. In contrast, significant toxicity was observed among patients treated with
erlotinib and temsirolimus in a phase I dose escalation study, such that de-escalation of study
agents was required. [65] An explanation for differences in tolerability between these regimens
may be due to different pharmacokinetic interactions or undefined pharmacogenetic factors.
We report limited efficacy among heavily pre-treated, recurrent GBM patients treated with
erlotinib and sirolimus. Due to limitations of the current study, it is not clear whether this
regimen is truly inactive or if our dosing schedule was inadequate to achieve intra-tumoral
concentrations sufficient to inhibit the intended PI3-AKT pathway targets. Although the
rationale for combinatorial regimens that simultaneously target key upstream and downstream
signal transduction pathway mediators is logical and supported preclinically, effective
translation of this concept into the clinic will require further evaluation of clinical, tumor
biomarker, pharmacokinetic and intra-tumoral pharmacodynamic measures.
Acknowledgments
This work was supported by NIH Grants NS20023 and CA11898; NIH Grant MO1 RR 30, GCRC Program, NCRR;
and NCI SPORE 1 P20 CA096890
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Abbreviations list
ANC Absolute neutrophil count
AST Aspartate aminotransferase
BBB Blood-brain barrier
BUN Blood urea nitrogen
CBC Complete blood count
CI Confidence intervals
CNS Central nervous system
CR Complete response
CTC Common Toxicity Criteria
CYP Cytochrome p450
DLT Dose-limiting toxicity
EGFR Epidermal growth factor receptor
EIAEDs Enzyme-inducing antieptileptic drugs
EGFR Epidermal growth factor receptor
GBM Glioblastoma multiforme
GS Gliosarcoma
IRB Institutional review board
ITT Intent-to treat
KPS Karnofsky performance status
MG Malignant glioma
MTD Maximum-tolerated dose
mTOR Mammalian target of rapamycin
NCI National Cancer Institute
NE Non-estimable
pAKT Phosphorylated akt murine thymomoa viral oncogene homologue 1
PD Progressive disease
PFS Progression-free survival
P-gp p-glycoprotein
pMAPK Phosphorylated mitogen activated protein kinase
PR Partial response
pS-6 Phosphorylated S-6 ribosomal protein
PTEN Phosphatase and tensin homologue
SD Stable disease
TKI Tyrosine kinase inhibitor
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VEGF Vascular endothelial growth factor
XRT External beam radiotherapy
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Fig. 1.
The immunohistochemical profile of patient A103 demonstrates a strong diffuse
immunoreactivity for EGFR wild type, b 30% reactivity for PTEN that correlates with PTEN
loss; c negative reactivity for EGFRvIII, d diffuse reactivity for pAKT, and e rare cells
exhibiting pS6 immunoreactivity with a positive internal neuronal control (arrow). The internal
bar represents 50 microns
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Fig. 2.
Kaplan–Meier plots of progression-free survival (Fig. 1a) and overall survival (Fig. 1b)
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Reardon et al. Page 18
Table 1
Patient demographics
Characteristic Not on EIAEDs
(n = 24) On EIAEDs
(n = 8) All
(n = 32)
Age (years)
Median 53 59 54
Range 40–71 45–66 40–71
Gender
Male 18 (75) 7 (88) 25 (78)
Female 6 (25) 1 (12) 7 (22)
KPS
90–100 12 (50) 6 (75) 18 (50)
80 8 (33) 1 (12) 7 (22)
70 4 (17) 1 (12) 5 (16)
Surgery prior to enrollment
GTR 0 1 (13) 1 (3)
STR 0 0 0
Biopsy 4 (13) 2 (25) 6 (19)
None 20 (63) 5 (63) 25 (78)
Prior treatment
XRT 24 (100) 8 (100) 32 (100)
No. prior chemotherapy
Rx agents
16 (25) 2 (25) 8 (25)
26 (25) 1 (12) 7 (22)
37 (29) 1 (12) 8 (25)
≥45 (21) 4 (50) 9 (28)
Time from original diagnosis (weeks)
Median 49.7 58 54.4
Range 19.6–158 31.9–153.9 19.6–158
Prior Bevacizumab
Yes 6 (25) 3 (38) 9 (28)
No 18 (75) 5 (62) 23 (72)
Number Prior PD
110 (42) 3 (38) 13 (41)
26 (25) 5 (62) 11 (34)
37 (29) 0 7 (22)
≥41 (4) 0 1 (3)
EIAEDs Enzyme-inducing anti-epileptic drug; GTR Gross total resection; KPS Karnofsky performance status; PD Progressive disease; STR Subtotal
resection; XRT External beam radiotherapy
Numbers in parentheses indicate percentage unless otherwise indicated
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Table 2
Number of patients with grade ≥2 adverse events
Toxicity grade 2 3 4
Stratum1A B A B A B
Number of patients 24 8 24 8 24 8
Anemia 1 (4) 0 0 0 0 0
Anorexia 2 (8) 1 (13) 0 0 0 0
Creatinine elevation 0 0 0 1 (13) 0 0
Diarrhea 5 (21) 5 (63) 0 0 0 0
Fatigue 3 (9) 4 (50) 2 (8) 0 0 0
Hypercholesterolemia 4 (17) 0 0 0 0 0
Hypertriglyceridemia 3 (13) 0 1 (4) 0 0 0
Hypoalbuminemia 3 (13) 0 0 0 0 0
Hypocalcemia 1 (4) 0 0 0 0 0
Hypokalemia 1 (4) 0 1 (4) 0 0 0
Hypophosphatemia 0 0 1 (4) 0 0 0
Infection 2 (8) 1 (13) 1 (4) 0 0 0
Mucositis 5 (21) 4 (50) 2 (8) 0 0 0
Nausea/emesis 2 (8) 1 (13) 0 0 0 0
Neutropenia 1 (4) 0 1 (4) 0 0 0
Rash 7 (29) 5 (63) 6 (25) 1 (13) 0 0
Thrombocytopenia 0 1 (13) 2 (8) 1 (13) 1 (4) 0
Transaminase elevation 0 0 0 1 (13) 0 0
Weight loss 1 (4) 0 0 0 0 0
Numbers in parentheses refer to percentages
1Stratum A: Patients not on CYP3A-inducing anti-epileptics (EIA-EDS) including phenytoin, carbamazepine, phenobarbitol, oxcarbazepine and primidone
Stratum B: Patients on EIAEDs
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Table 3
Outcome
Total # Failed Median (weeks) 6 Months Survival (%) 1 Year survival (%) Logrank_p
Overall survival
All Patients 32 27 33.8 (21.9, 53.6) 59.4 (40.5, 74) 34.4 (18.8, 50.6)
Strata
Non-EIAEDS 24 19 32.5 (20, 57.6) 58.3% (36.4, 75) 33.3 (15.9, 51.9) 0.43
EIAEDS 8 8 33.8 (15.4, 55.4) 62.5% (22.9, 86.1) 37.5 (8.7, 67.4)
Progression free survival
All patients 32 32 6.9 (3.9, 11) 3.1% (0.2, 13.7)
Strata
Non-EIAEDS 24 24 8.4 (3.9, 12.1) 4.2% (0.3, 17.6) 0.04
EIAEDS 8 8 4 (3.9, 7.4) 0
Prognostic factors
Adverse Event
Grade 2 or higher rash No 15 15 6.7 (4, 11.1) 0 0.64
Yes 17 17 7.3 (3.9, 11.9) 5.9 (0.4, 23.5)
Grade 3 rash No 25 25 6.7 (3.9, 11) 4 (0.3, 17) 0.59
Yes 7 7 7.3 (3.9, 15.9) 0
Diarrhea No 23 23 6.7 (4, 11.9) 4.3% (0.3, 18.2) 0.59
Yes 9 9 7.3 (3.9, 9.4) 0
Hyperlipidemia No 28 28 5.4 (3.9, 7.7) 0 0.029
Yes 4 4 16.7 (3.6, 39.9) 25 (0.9, 66.5)
Tumor marker
EGFR-FISH Polysomy 9 9 7.7 (3.9, 12.1) 11.1% (0.6, 38.8) 0.49
Amplified 4 4 5.7 (3.6, 12.7) 0
EGFR-IHC 30%–60% pos. 4 4 3.9 (2.4, 7.7) 0 0.35
80%–90% pos 11 11 4 (3.9, 9.4) 0
EGFR-IHC <90% pos. 8 8 4 (3.9, 7.7) 0 0.64
≥90% pos. 7 7 3.9 (3.6, 11.1) 0
PTEN-FISH Intact 11 11 4 (3.9, 11.9) 0 0.60
Loss 11 11 7.4 (3.9, 12.7) 9.1 (0.5, 33.3)
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Total # Failed Median (weeks) 6 Months Survival (%) 1 Year survival (%) Logrank_p
PTEN-IHC Intact 8 8 4 (3.6, 12.7) 0 0.23
Loss 12 12 10.3 (7, 12.1) 8.3 (0.5, 31.1)
EGFRvIII-IHC Negative 16 16 8.6 (4, 12.1) 6.3 (0.4, 24.7) 0.36
Positive 4 4 5.5 (3.6, 12.7) 0
pAKT-IHC Negative 9 9 4 (3.6, 7.4) 0 0.04
Positive 7 7 9.4 (4, 12.7) 14.3 (0.7, 46.5)
pS6-IHC Negative 2 2 23.6 (7.4, 39.9) 50 (0.6, 91) 0.18
Positive 10 10 5.5 (3.9, 9.4) 0
pMAPK-IHC Negative 3 3 4 (4, 7.7) 0 0.49
Positive 2 2 5.5 (3.6, 7.4) 0
Numbers in parentheses refer to 95% confidence intervals unless otherwise indicated
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