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David Akhavan,
Alexandra L Pourzia,
Alex A Nourian,
Kevin J Williams,
David Nathanson,
Ivan Babic,
Genaro R Villa,
Kazuhiro Tanaka,
Ali Nael,
Huijun Yang, [......],
William H Yong,
Mitchell Flagg,
Fuyuhiko Tamanoi,
Takashi Sasayama,
C David James,
Harley I Kornblum,
Timothy F Cloughesy,
Webster K Cavenee,
Steven J Bensinger,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here we demonstrate the first example of this alternate mechanism in brain tumors by showing that EGFR-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing PDGFRβ. Mechanistic studies demonstrate that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1 and ERK-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, non-genetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy.
Cancer discovery. 03/2013;
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Akio Iwanami,
Beatrice Gini,
Ciro Zanca,
Tomoo Matsutani,
Alvaro Assuncao,
Ali Nael, Julie Dang,
Huijun Yang,
Shaojun Zhu,
Jun Kohyama,
Issay Kitabayashi,
Webster K Cavenee,
Timothy F Cloughesy,
Frank B Furnari,
Masaya Nakamura,
Yoshiaki Toyama,
Hideyuki Okano,
Paul S Mischel
[show abstract]
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ABSTRACT: Despite their nearly universal activation of mammalian target of rapamycin (mTOR) signaling, glioblastomas (GBMs) are strikingly resistant to mTOR-targeted therapy. We analyzed GBM cell lines, patient-derived tumor cell cultures, and clinical samples from patients in phase 1 clinical trials, and find that the promyelocytic leukemia (PML) gene mediates resistance to mTOR-targeted therapies. Direct mTOR inhibitors and EGF receptor (EGFR) inhibitors that block downstream mTOR signaling promote nuclear PML expression in GBMs, and genetic overexpression and knockdown approaches demonstrate that PML prevents mTOR and EGFR inhibitor-dependent cell death. Low doses of the PML inhibitor, arsenic trioxide, abrogate PML expression and reverse mTOR kinase inhibitor resistance in vivo, thus markedly inhibiting tumor growth and promoting tumor cell death in mice. These results identify a unique role for PML in mTOR and EGFR inhibitor resistance and provide a strong rationale for a combination therapeutic strategy to overcome it.
Proceedings of the National Academy of Sciences 02/2013; · 9.68 Impact Factor
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Tim R Fenton,
David Nathanson,
Claudio Ponte de Albuquerque,
Daisuke Kuga,
Akio Iwanami, Julie Dang,
Huijun Yang,
Kazuhiro Tanaka,
Sueli Mieko Oba-Shinjo,
Miyuki Uno, [......],
Jill Wykosky,
Robert M Bachoo,
C David James,
Ronald A DePinho,
Scott R Vandenberg,
Huilin Zhou,
Suely K N Marie,
Paul S Mischel,
Webster K Cavenee,
Frank B Furnari
[show abstract]
[hide abstract]
ABSTRACT: Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.
Proceedings of the National Academy of Sciences 08/2012; 109(35):14164-9. · 9.68 Impact Factor
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Igor Vivanco,
H Ian Robins,
Daniel Rohle,
Carl Campos,
Christian Grommes,
Phioanh Leia Nghiemphu,
Sara Kubek,
Barbara Oldrini,
Milan G Chheda,
Nicolas Yannuzzi, [......],
Steve Horvath,
Nian Wu,
Andrew B Lassman,
Lisa M DeAngelis,
William H Yong,
John G Kuhn,
Paul S Mischel,
Minesh P Mehta,
Timothy F Cloughesy,
Ingo K Mellinghoff
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cancer discovery. 05/2012; 2(5):458-71.
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[show abstract]
[hide abstract]
ABSTRACT: PTEN-controlled PI3K-AKT-mTOR pathway represents one of the most deregulated signaling pathways in human cancers. With many small molecule inhibitors that target PI3K-AKT-mTOR pathway being exploited clinically, sensitive and reliable ways of stratifying patients according to their PTEN functional status and determining treatment outcomes are urgently needed. Heterogeneous loss of PTEN is commonly associated with human cancers and yet PTEN can also be regulated on epigenetic, transcriptional or post-translational levels, which makes the use of simple protein or gene expression-based analyses in determining PTEN status less accurate. In this study, we used network component analysis to identify 20 transcription factors (TFs) whose activities deduced from their target gene expressions were immediately altered upon the re-expression of PTEN in a PTEN-inducible system. Interestingly, PTEN controls the activities (TFA) rather than the expression levels of majority of these TFs and these PTEN-controlled TFAs are substantially altered in prostate cancer mouse models. Importantly, the activities of these TFs can be used to predict PTEN status in human prostate, breast and brain tumor samples with enhanced reliability when compared to straightforward IHC-based or expression-based analysis. Furthermore, our analysis indicates that unique sets of PTEN-controlled TFAs significantly contribute to specific tumor types. Together, our findings reveal that TFAs may be used as "signatures" for predicting PTEN functional status and elucidate the transcriptional architectures underlying human cancers caused by PTEN loss.
PLoS ONE 01/2012; 7(2):e31053. · 4.09 Impact Factor
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Kazuhiro Tanaka,
Ivan Babic,
David Nathanson,
David Akhavan,
Deliang Guo,
Beatrice Gini, Julie Dang,
Shaojun Zhu,
Huijun Yang,
Jason De Jesus, [......],
Hancai Dan,
Amanda Rinkenbaugh,
William H Yong,
Harry V Vinters,
Joseph F Gera,
Webster K Cavenee,
Timothy F Cloughesy,
Brendan D Manning,
Albert S Baldwin,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.
Cancer discovery. 11/2011; 1(6):524-38.
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Deliang Guo,
Felicia Reinitz,
Mary Youssef,
Cynthia Hong,
David Nathanson,
David Akhavan,
Daisuke Kuga,
Ali Nael Amzajerdi,
Horacio Soto,
Shaojun Zhu, [......],
H Ian Robins,
Michael D Prados,
Lisa M Deangelis,
Ingo K Mellinghoff,
Minesh P Mehta,
C David James,
Arnab Chakravarti,
Timothy F Cloughesy,
Peter Tontonoz,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.
Cancer discovery. 09/2011; 1(5):442-56.
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Igor Vivanco,
Daniel Rohle,
Matthias Versele,
Akio Iwanami,
Daisuke Kuga,
Barbara Oldrini,
Kazuhiro Tanaka, Julie Dang,
Sara Kubek,
Nicolaos Palaskas, [......],
Sigrid Rajasekaran,
Ayyappan Rajasekaran,
Linda M Liau,
Timothy F Cloughesy,
Ivan Dikic,
Cameron Brennan,
Hong Wu,
Paul S Mischel,
Timothy Perera,
Ingo K Mellinghoff
[show abstract]
[hide abstract]
ABSTRACT: The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their "dependence" on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.
Proceedings of the National Academy of Sciences 03/2010; 107(14):6459-64. · 9.68 Impact Factor
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Caroline Gregorian,
Jonathan Nakashima,
Sarah M Dry,
P Leia Nghiemphu,
Kate Barzan Smith,
Yan Ao, Julie Dang,
Gregory Lawson,
Ingo K Mellinghoff,
Paul S Mischel,
Michael Phelps,
Luis F Parada,
Xin Liu,
Michael V Sofroniew,
Fritz C Eilber,
Hong Wu
[show abstract]
[hide abstract]
ABSTRACT: Patients with neurofibromatosis type 1 (NF1) carry approximately a 10% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST). Although the molecular mechanisms underlying NF1 to MPNST malignant transformation remain unclear, alterations of both the RAS/RAF/MAPK and PI3K/AKT/mTOR signaling pathways have been implicated. In a series of genetically engineered murine models, we perturbed RAS/RAF/MAPK or/and PTEN/PI3K/AKT pathway, individually or simultaneously, via conditional activation of K-ras oncogene or deletion of Nf1 or Pten tumor suppressor genes. Only K-Ras activation in combination with a single Pten allele deletion led to 100% penetrable development of NF lesions and subsequent progression to MPNST. Importantly, loss or decrease in PTEN expression was found in all murine MPNSTs and a majority of human NF1-associated MPNST lesions, suggesting that PTEN dosage and its controlled signaling pathways are critical for transformation of NFs to MPNST. Using noninvasive in vivo PET-CT imaging, we demonstrated that FDG can be used to identify the malignant transformation in both murine and human MPNSTs. Our data suggest that combined inhibition of RAS/RAF/MAPK and PTEN/PI3K/AKT pathways may be beneficial for patients with MPNST.
Proceedings of the National Academy of Sciences 10/2009; 106(46):19479-84. · 9.68 Impact Factor
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Kan V Lu,
Shaojun Zhu,
Anna Cvrljevic,
Tiffany T Huang,
Shawn Sarkaria,
David Ahkavan, Julie Dang,
Eduard B Dinca,
Seema B Plaisier,
Isaac Oderberg, [......],
Roberto Weinmann,
Timothy F Cloughesy,
Stanley F Nelson,
Gabriele Bergers,
Thomas Graeber,
Frank B Furnari,
C David James,
Webster K Cavenee,
Terrance G Johns,
Paul S Mischel
[show abstract]
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ABSTRACT: Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.
Cancer Research 09/2009; 69(17):6889-98. · 7.86 Impact Factor
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Deliang Guo,
Isabel J Hildebrandt,
Robert M Prins,
Horacio Soto,
Mary M Mazzotta, Julie Dang,
Johannes Czernin,
John Y-J Shyy,
Andrew D Watson,
Michael Phelps,
Caius G Radu,
Timothy F Cloughesy,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: The EGFR/PI3K/Akt/mTOR signaling pathway is activated in many cancers including glioblastoma, yet mTOR inhibitors have largely failed to show efficacy in the clinic. Rapamycin promotes feedback activation of Akt in some patients, potentially underlying clinical resistance and raising the need for alternative approaches to block mTOR signaling. AMPK is a metabolic checkpoint that integrates growth factor signaling with cellular metabolism, in part by negatively regulating mTOR. We used pharmacological and genetic approaches to determine whether AMPK activation could block glioblastoma growth and cellular metabolism, and we examined the contribution of EGFR signaling in determining response in vitro and in vivo. The AMPK-agonist AICAR, and activated AMPK adenovirus, inhibited mTOR signaling and blocked the growth of glioblastoma cells expressing the activated EGFR mutant, EGFRvIII. Across a spectrum of EGFR-activated cancer cell lines, AICAR was more effective than rapamycin at blocking tumor cell proliferation, despite less efficient inhibition of mTORC1 signaling. Unexpectedly, addition of the metabolic products of cholesterol and fatty acid synthesis rescued the growth inhibitory effect of AICAR, whereas inhibition of these lipogenic enzymes mimicked AMPK activation, thus demonstrating that AMPK blocked tumor cell proliferation primarily through inhibition of cholesterol and fatty acid synthesis. Most importantly, AICAR treatment in mice significantly inhibited the growth and glycolysis (as measured by (18)fluoro-2-deoxyglucose microPET) of glioblastoma xenografts engineered to express EGFRvIII, but not their parental counterparts. These results suggest a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.
Proceedings of the National Academy of Sciences 08/2009; 106(31):12932-7. · 9.68 Impact Factor
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Deliang Guo,
Robert M Prins, Julie Dang,
Daisuke Kuga,
Akio Iwanami,
Horacio Soto,
Kelly Y Lin,
Tiffany T Huang,
David Akhavan,
M Benjamin Hock, [......],
Andrew D Watson,
John G Kuhn,
H Ian Robins,
Minesh P Mehta,
Patrick Y Wen,
Lisa M DeAngelis,
Michael D Prados,
Ingo K Mellinghoff,
Timothy F Cloughesy,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin's poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.
Science Signaling 01/2009; 2(101):ra82. · 7.50 Impact Factor
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Peter T Jindra,
Aileen Hsueh,
Longshen Hong,
David Gjertson,
Xiu-Da Shen,
Feng Gao, Julie Dang,
Paul S Mischel,
William M Baldwin,
Michael C Fishbein,
Jerzy W Kupiec-Weglinski,
Elaine F Reed
[show abstract]
[hide abstract]
ABSTRACT: Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2K(b)/D(b)) received a fully MHC-incompatible BALB/c (H-2K(d)/D(d)) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2K(d) Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.
The Journal of Immunology 03/2008; 180(4):2214-24. · 5.79 Impact Factor
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Koji Yoshimoto, Julie Dang,
Shaojun Zhu,
David Nathanson,
Tiffany Huang,
Rebecca Dumont,
David B Seligson,
William H Yong,
Zhenggang Xiong,
Nagesh Rao,
Henrik Winther,
Arnab Chakravarti,
Darell D Bigner,
Ingo K Mellinghoff,
Steve Horvath,
Webster K Cavenee,
Timothy F Cloughesy,
Paul S Mischel
[show abstract]
[hide abstract]
ABSTRACT: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge.
We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization.
The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00).
This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.
Clinical Cancer Research 02/2008; 14(2):488-93. · 7.74 Impact Factor
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Tim F Cloughesy,
Koji Yoshimoto,
Phioanh Nghiemphu,
Kevin Brown, Julie Dang,
Shaojun Zhu,
Teli Hsueh,
Yinan Chen,
Wei Wang,
David Youngkin, [......],
Marvin Bergsneider,
Albert Lai,
Richard Green,
Tom Oglesby,
Michael Koleto,
Jeff Trent,
Steve Horvath,
Paul S Mischel,
Ingo K Mellinghoff,
Charles L Sawyers
[show abstract]
[hide abstract]
ABSTRACT: 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.
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).
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.
http://www.ClinicalTrials.gov (#NCT00047073).
PLoS Medicine 01/2008; 5(1):e8. · 16.27 Impact Factor
