Phioanh L. Nghiemphu

Antitumor activity of rapamycin in a phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population. Methods and Findings Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test). Conclusions Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development. Trial registration: http://www.ClinicalTrials.gov (#NCT00047073).

Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme

PURPOSE This open-label, prospective, multicenter single-arm phase II study combined bevacizumab (BV) with radiation therapy (RT) and temozolomide (TMZ) for the treatment of newly diagnosed glioblastoma (GBM). The objectives were to determine the efficacy of this treatment combination and the associated toxicity. PATIENTS AND METHODS Seventy patients with newly diagnosed GBM were enrolled between August 2006 and November 2008. Patients received standard RT starting within 3 to 6 weeks after surgery with concurrent administration of daily TMZ and biweekly BV. After completion of RT, patients resumed TMZ for 5 days every 4 weeks and continued biweekly BV. MGMT promoter methylation was assessed on patient tumor tissue. A University of California, Los Angeles/Kaiser Permanente Los Angeles (KPLA) control cohort of newly diagnosed patients treated with first-line RT and TMZ who had mostly received BV at recurrence was derived for comparison. RESULTS The overall survival (OS) and progression-free survival (PFS) were 19.6 and 13.6 months, respectively, compared to 21.1 and 7.6 months in the University of California, Los Angeles/KPLA control cohort, and 14.6 and 6.9 months in the European Organisation for Research and Treatment of Cancer-National Cancer Institute of Canada cohort. Correlation of MGMT promoter methylation and improved OS and PFS was retained in the study group. Comparative subset analysis showed that poor prognosis patients (recursive partitioning analysis class V/VI) may derive an early benefit from the use of first-line BV. Toxicity attributable to RT/TMZ was similar, and additional toxicities were consistent with those reported in other BV trials. CONCLUSION Patients treated with BV and TMZ during and after RT showed improved PFS without improved OS compared to the University of California, Los Angeles/KPLA control group. Additional studies are warranted to determine if BV administered first-line improves survival compared to BV at recurrence.

MRI in patients with high-grade gliomas treated with bevacizumab and chemotherapy

To the Editor: Pope et al. retrospectively reviewed 14 patients with recurrent high-grade gliomas (HGG) treated with chemotherapy and bevacizumab.1 Fifty percent of patients responded to treatment determined by post-treatment MRI. Several aspects of this report merit commentary. All patients in the study previously failed primary therapy and one prior salvage therapy. The number of patients treated within 3 months of radiotherapy was unclear. Though the protocol specified at least 4 weeks elapsed since administration of radiotherapy, it is recognized that early delayed radiotherapy changes seen on MRI may simulate tumor progression and resolve over time.2 The performance of MRI to assess response to therapy in patients with recurrent HGG is customarily performed every 2 months and coordinated with chemotherapy cycles. In this report, first post-treatment MRI appears desynchronized with chemotherapy administration. As response rates were based on first post-treatment MRI, response appears not to be bevacizumab dose dependent. To this author’s knowledge, there has not been a single agent trial of bevacizumab for recurrent HGG though trials in other solid cancers indicate a less than 10% single agent response rate. The rationale for combining bevacizumab with CPT-11 was not mentioned in this article. A number of reports evaluating CPT-11 as a single agent for recurrent glioblastoma have concluded the agent has little efficacy.3 The first report by Stark-Vance indicating response to the combination of bevacizumab and CPT-11 for recurrent HGG was not cited.4 The durability of response was also not reported. Clinical trials in recurrent HGG report response duration by overall survival, time to tumor progression, and 6-month progression free survival. Fifteen percent 6-month progression free survival for patients with recurrent glioblastoma is used as the standard by which new trials are compared with respect to efficacy.5 A variety of methods have been utilized to assess response to anti-angiogenic agents including dynamic contrast enhanced MRI. These methods provide proof of principle by quantifying tumor blood volume or perfusion. Pope et al. do not report whether the observed response represents an anti-angiogenic effect on tumor vasculature or an effect on blood brain barrier disruption with normalization of peritumoral edema and diminished tumor contrast enhancement. Pope et al emphasize the complexity of treating recurrent HGG and remind neurologists that the approach to malignant gliomas continues to evolve and will increasingly utilize targeted therapies such as antiangiogenic agents.

Recurrent Glioblastoma Multiforme: ADC Histogram Analysis Predicts Response to Bevacizumab Treatment

PURPOSE To determine if apparent diffusion coefficient (ADC) histogram analysis can stratify progression-free survival in patients with recurrent glioblastoma multiforme (GBM) prior to bevacizumab treatment. MATERIALS AND METHODS The study was approved by the institutional review board and was HIPAA compliant; informed consent was obtained. Bevacizumab-treated and control patients (41 per cohort) diagnosed with recurrent GBM were analyzed by using whole enhancing tumor ADC histograms with a two normal distribution mixture fitting curve on baseline (pretreatment) magnetic resonance (MR) images to generate ADC classifiers, including the overall mean ADC as well as the mean ADC from the lower curve (ADC(L)). Overall and 6-month progression-free survival (as defined by the Macdonald criteria) was determined by using Cox proportional hazard ratios and the Kaplan-Meier method with log-rank test. RESULTS For bevacizumab-treated patients, the hazard ratio for progression by 6 months in patients with less than versus greater than mean ADC(L) was 4.1 (95% confidence interval: 1.6, 10.4), and there was a 2.75-fold reduction in the median time to progression. For the control patients, there was no significant difference in median time to progression for the patients with low versus high ADC(L) (hazard ratio, 1.8; 95% confidence interval: 0.9, 3.7). For bevacizumab-treated patients, pretreatment ADC more accurately stratified 6-month progression-free survival than did change in enhancing tumor volume at first follow-up (73% vs 58% accuracy, P = .034). CONCLUSION Pretreatment ADC histogram analysis can stratify progression-free survival in bevacizumab-treated patients with recurrent GBM.

Evidence for Sequenced Molecular Evolution of IDH1 Mutant Glioblastoma From a Distinct Cell of Origin

PURPOSE Mutation in isocitrate dehydrogenase 1 (IDH1) at R132 (IDH1(R132MUT)) is frequent in low-grade diffuse gliomas and, within glioblastoma (GBM), has been proposed as a marker for GBMs that arise by transformation from lower-grade gliomas, regardless of clinical history. To determine how GBMs arising with IDH1(R132MUT) differ from other GBMs, we undertook a comprehensive comparison of patients presenting clinically with primary GBM as a function of IDH1(R132) mutation status. PATIENTS AND METHODS In all, 618 treatment-naive primary GBMs and 235 lower-grade diffuse gliomas were sequenced for IDH1(R132) and analyzed for demographic, radiographic, anatomic, histologic, genomic, epigenetic, and transcriptional characteristics. RESULTS Investigation revealed a constellation of features that distinguishes IDH1(R132MUT) GBMs from other GBMs (including frontal location and lesser extent of contrast enhancement and necrosis), relates them to lower-grade IDH1(R132MUT) gliomas, and supports the concept that IDH1(R132MUT) gliomas arise from a neural precursor population that is spatially and temporally restricted in the brain. The observed patterns of DNA sequence, methylation, and copy number alterations support a model of ordered molecular evolution of IDH1(R132MUT) GBM in which the appearance of mutant IDH1 protein is an initial event, followed by production of p53 mutant protein, and finally by copy number alterations of PTEN and EGFR. CONCLUSION Although histologically similar, GBMs arising with and without IDH1(R132MUT) appear to represent distinct disease entities that arise from separate cell types of origin as the result of largely nonoverlapping sets of molecular events. Optimal clinical management should account for the distinction between these GBM disease subtypes.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

Bevacizumab and chemotherapy for recurrent glioblastoma A single-institution experience

Objective: Bevacizumab has been shown to be effective in the treatment of recurrent glioblastoma in combination with chemotherapy compared with historic controls but not in randomized trials. Methods: We conducted a retrospective analysis of patients treated for recurrent glioblastoma with bevacizumab vs a control group of patients, comparing progression-free survival (PFS) and overall survival (OS) between the two groups, and performed subgroup analysis based on age and performance status. Expression of vascular endothelial growth factor (VEGF) based on age was examined using DNA microarray analysis. We also evaluated the impact of bevacizumab on quality of life. Results: We identified 44 patients who received bevacizumab and 79 patients who had not been treated with bevacizumab. There was a significant improvement in PFS and OS in the bevacizumab-treated group. Patients of older age (≥55 years) and poor performance status (Karnofsky Performance Status ≤80) had significantly better PFS when treated with bevacizumab, and bevacizumab-treated older patients had significantly increased OS. VEGF expression was significantly higher in older glioblastoma patients (aged ≥55 years). Patients treated with bevacizumab also required less dexamethasone use and maintained their functional status longer than the control group. Conclusions: Bevacizumab in combination with chemotherapy may be a more effective treatment for recurrent glioblastoma and warrants further randomized prospective studies to determine its effect on survival. Bevacizumab also has more effect in those with older age and might reflect biologic differences in glioblastoma in different age groups as seen with the expression of vascular endothelial growth factor. GBM = glioblastoma; HR = hazard ratio; KPS = Karnofsky Performance Status; OS = overall survival; PFS = progression-free survival; VEGF = vascular endothelial growth factor.

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase–activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome–wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

Phase II Pilot Study of Bevacizumab in Combination with Temozolomide and Regional Radiation Therapy for Up-Front Treatment of Patients With Newly Diagnosed Glioblastoma Multiforme: Interim Analysis of Safety and Tolerability

PURPOSE To assess interim safety and tolerability of a 10-patient, Phase II pilot study using bevacizumab (BV) in combination with temozolomide (TMZ) and regional radiation therapy (RT) in the up-front treatment of patients with newly diagnosed glioblastoma. METHODS AND MATERIALS All patients received standard external beam regional RT of 60.0 Gy in 30 fractions started within 3 to 5 weeks after surgery. Concurrently TMZ was given daily at 75 mg/m(2) for 42 days during RT, and BV was given every 2 weeks at 10 mg/kg starting with the first day of RT/TMZ. After a 2-week interval upon completion of RT, the post-RT phase commenced with resumption of TMZ at 150 to 200 mg/m(2) for 5 days every 4 weeks and continuation of BV every 2 weeks. RESULTS For these 10 patients, toxicities were compiled until study discontinuation or up to approximately 40 weeks from initial study treatment for those remaining on-study. In terms of serious immediate or delayed neurotoxicity, 1 patient developed presumed radiation-induced optic neuropathy. Among the toxicities that could be potentially treatment related, relatively high incidences of fatigue, myelotoxicity, wound breakdown, and deep venous thrombosis/pulmonary embolism were observed. CONCLUSION The observed toxicities were acceptable to continue enrollment toward the overall target group of 70 patients. Preliminary efficacy analysis shows encouraging mean progression-free survival. At this time data are not sufficient to encourage routine off-label use of BV combined with TMZ/RT in the setting of newly diagnosed glioblastoma without longer follow-up, enrollment of additional patients, and thorough efficacy assessment.

Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to EGFR kinase inhibitors.

UNLABELLED Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.