EGFR amplified and overexpressing glioblastomas and association with better response to adjuvant metronomic temozolomide.
BACKGROUND
Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment.


METHODS
We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student's t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided.


RESULTS
No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (P interaction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients.


CONCLUSIONS
EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide-based therapies.
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Manuela
M
Cominelli
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Salvatore
S
Grisanti
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Stefania
S
Mazzoleni
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Caterina
C
Branca
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Luciano
L
Buttolo
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Daniela
D
Furlan
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Barbara
B
Liserre
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Maria Fausta
MF
Bonetti
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Daniela
D
Medicina
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Vilma
V
Pellegrini
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Michela
M
Buglione
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Roberto
R
Liserre
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Serena
S
Pellegatta
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Gaetano
G
Finocchiaro
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Piero
P
Dalerba
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Fabio
F
Facchetti
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Marina
M
Pizzi
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Rossella
R
Galli
Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).
Pietro Luigi
PL
Poliani
D059250:Administration, Metronomic; D000328:Adult; D000704:Analysis of Variance; D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage / Q000627:therapeutic use*; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery; D017024:Chemotherapy, Adjuvant; D003606:Dacarbazine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000627:therapeutic use; D018572:Disease-Free Survival; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008875:Middle Aged; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D016017:Odds Ratio; D016016:Proportional Hazards Models; D000077204:Temozolomide; D015854:Up-Regulation
D018906:Antineoplastic Agents, Alkylating; D003606:Dacarbazine; C512478:EGFR protein, human; D066246:ErbB Receptors; D000077204:Temozolomide