Gregory W. Canute

Activity of anti-epidermal growth factor receptor monoclonal antibody C225 against glioblastoma multiforme

OBJECTIVE Overexpression of epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) secondary to EGFR gene amplification is associated with a more aggressive tumor phenotype and a worse clinical outcome. The purpose of this study was to analyze whether blocking this receptor with the anti-EGFR chimeric monoclonal antibody C225 would decrease proliferation and increase apoptosis in GBM cells. METHODS EGFR expression and amplification were analyzed for seven human GBM cell lines. These lines were then exposed to different concentrations of C225 for 48 hours, 72 hours, and 7 days, after which time cytotoxicity, apoptosis, and vascular endothelial growth factor expression were assessed in vitro. Two EGFR-amplified human GBM were implanted in the flanks of nude mice, and the animals received C225 twice per week intraperitoneally for 5 weeks. Tumor volumes and survival times were compared with those of sham-treated mice. RESULTS EGFR gene amplification was demonstrated in three of the primary GBM lines. C225 treatment produced significant cytotoxicity in all three EGFR-amplified GBM lines, but not in unamplified lines. Flow cytometry demonstrated increased apoptosis in C225-treated, EGFR-amplified GBM lines, but not in unamplified lines. There was a decrease in vascular endothelial growth factor expression in all GBM lines with exposure to C225. Tumor-bearing mice treated with C225 experienced significant inhibition of tumor growth as well as a 200% increase in median survival. CONCLUSION Blocking EGFR in GBM cells that overexpress this receptor significantly changes tumor cell biology by promoting apoptosis while decreasing proliferation and vascular endothelial growth factor expression. This approach holds great promise for the treatment of patients with GBMs.

Anti-epidermal growth factor receptor monoclonal antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo

OBJECTIVE:Previously, we demonstrated that the anti-epidermal growth factor receptor (EGFR) antibody cetuximab alone was effective against EGFR-amplified glioblastoma multiforme (GBM) cells in vivo and in vitro. The purpose of the present work was to study further the effectiveness of cetuximab as a monotherapy as well as combining it with radiation therapy or chemotherapy. METHODS:EGFR-amplified GBM cells were implanted either in the flanks of nude mice to determine the effectiveness of cetuximab on larger tumor burden or intracranially to assess the ability of cetuximab to cross the blood-brain barrier. Cells were also exposed to cetuximab in combination with radiation in vivo or chemotherapeutic agents in vitro. RESULTS:Increasing tumor burden in the flanks of mice decreased the amount of tumor growth inhibition. For the first two intracranial models using cetuximab for 5 weeks, the treated mice had a significant increase in median survival compared with controls. When cetuximab was given indefinitely, the results were encouraging, with an increase in median survival for the treated group not yet reached but at least 900%. Mice with flank GBM exposed to cetuximab and radiation had a larger increase in median survival than those with either treatment alone. Preliminary in vitro experiments using cetuximab and chemotherapeutic agents showed increased cytotoxicity. CONCLUSION:These results were encouraging, demonstrating the effectiveness of cetuximab against EGFR-amplified GBM. Surprisingly, cetuximab was effective when administered systemically in an intracranial model. Radiation augmented the effect of cetuximab on GBM in vitro and in vivo. In vitro analysis demonstrated additive effects for chemotherapeutic agents as well. These results confirm EGFR blockade with cetuximab as a potential treatment against human GBM.

Gamma Knife surgery in the management of radioresistant brain metastases in high-risk patients with melanoma, renal cell carcinoma, and sarcoma

OBJECT The purpose of this study was to examine the results of using Gamma Knife surgery (GKS) for brain metastases from classically radioresistant malignancies. METHODS The authors retrospectively reviewed the records of 76 patients with melanoma (50 patients), renal cell carcinoma (RCC; 23 patients), or sarcoma (3 patients) who underwent GKS between August 1998 and July 2007. Overall patient survival, intracranial progression, and local progression of individual lesions were analyzed. RESULTS The median age of the patients was 57 years (range 18-85 years) and median Karnofsky Performance Scale (KPS) score was 80 (range 20-100). Sixty-two patients (81.6%) had uncontrolled extracranial disease. A total of 303 intracranial lesions (average 3.97 per patient, range 1-27 lesions) were treated using GKS. More than 3 lesions were treated in 30 patients (39.5%). Median GKS tumor margin dose was 18 Gy (range 8-30 Gy). Thirty-seven patients (48.7%) underwent whole brain radiation therapy. The actuarial 12-month rate for freedom from local progression for individual lesions was 77.7% and was significantly higher for RCC compared with melanoma (93.6 vs 63.0%; p = 0.001). The percentage of coverage of the prescribed dose to target volume was the only treatment-related variable associated with local control: 12-month actuarial rate of freedom from local progression was 71.4% for lesions receiving >or= 90% coverage versus 0.0% for lesions receiving < 90% (p = 0.00048). Median overall survival was 5.1 months after GKS and 8.4 months after the discovery of brain metastases. Univariate analysis revealed that KPS score (p = 0.000004), recursive partitioning analysis class (p = 0.00043), and single metastases (p = 0.028), but not more than 3 metastases, to be prognostic factors of overall survival. The KPS score remained significant after multivariate analysis. Overall survival for patients with a KPS score >or= 70 was 7.1 months compared with 1.3 months for a KPS score <or= 60 (p = 0.013). CONCLUSIONS Gamma Knife surgery is an effective treatment option for patients with radioresistant brain metastases. In this setting, KPS score appeared to be a more important factor in predicting survival than having > 3 metastases. Higher rates of local tumor control were achieved for RCC in comparison with melanoma, and this may have an effect on survival in some patients. Although outcomes generally remained poor in this study population, these results suggest that GKS can be considered as a treatment option for many patients with radioresistant brain metastases, even if these patients have multiple lesions.

Soluble Fas-ligand (sFasL) in human astrocytoma cyst fluid is cytotoxic to T-cells : another potential means of immune evasion

Gliomas of all grades have been shown to express FasL, an apoptosis-inducing protein. Because of the ability of FasL to be cleaved from cell surfaces by metalloproteinases, soluble FasL can be released by FasL bearing cells into surrounding tissues. In the present study, we demonstrate the presence of sFasL in the cyst fluids of astrocytomas. Additionally, a human T-cell line, Jurkat, exposed to astrocytoma cyst fluid resulted in significantly increased cytotoxicity as compared to controls, an effect blocked by FasL neutralizing antibodies. This suggests that sFasL, may be utilized as a means of escaping immune surveillance by these tumors.

Apoptosis and Survival in High-grade Astrocytomas as Related to Tumor Fas (APO-1/CD95) Expression

Although a majority of high-grade gliomas express the apoptosis-inducing receptor Fas, little is known about the extent of apoptosis or prognostic significance of Fas expression in these tumors. In situ labeling of apoptotic cells and Ki-67 immunohistochemistry were performed on 51 high-grade human astrocytomas previously characterized for Fas expression. Survival data was compiled from patient records and correlated with tumor grade, apoptotic index (AI) and Fas expression. A significant correlation was found between tumor grade and the AI and Ki-67 labeling index (LI); however, only the AI increased significantly with Fas expression. The AI increased from 0.39 ± 0.12% to 0.82 ± 0.10% in grade III vs. IV astrocytomas (P = 0.003). The Ki-67-LI increased from 3.64 ± 1.5% to 11.35 ± 2.1% in grade III vs. IV astrocytomas (P = 0.004). Additionally, tumors expressing higher Fas levels had a greater AI than those expressing lower levels (0.81 ± 0.11% vs. 0.43 ± 0.11%) (P = 0.017). Despite longer median survivals for patients with tumors exhibiting high Fas expression, statistical significance was not achieved. Patients with grade III astrocytomas demonstrated a median survival of 20 vs. 18 months for tumors with high vs. low Fas expression (P = 0.51). Patients with grade IV astrocytomas demonstrated a median survival of 9 vs. 7.4 months for tumors with high vs. low Fas expression, respectively (P = 0.77). Although the degree of Fas expression in high-grade astrocytomas appears to correlate with the apoptotic rate, no overall differences in survival could be demonstrated between tumors expressing high vs. low Fas levels.

Neuromonitoring with pulse-train stimulation for implantation of thoracic pedicle screws: a blinded and randomized clinical study. Part 1. Methods and alarm criteria

OBJECT Reports of the accuracy of existing neuromonitoring methods for detecting or preventing medial malpositioning of thoracic pedicle screws have varied widely in their claimed effectiveness. The object of this study was to develop, test, and validate a novel neuromonitoring method for preventing medial malpositioning of pedicle screws in the thoracic spine during surgery. METHODS This is a prospective, blinded and randomized study using a novel combination of input (4-pulse stimulus trains delivered within the pedicle track) and output (evoked electromyography from leg muscles) to detect pedicle track trajectories that-once implanted with a screw-would cause that screw to breach the pedicles medial wall and encroach upon the spinal canal. For comparison, the authors also used screw stimulation as an input and evoked electromyogram from intercostal and abdominal muscles as output measures. Intraoperative electrophysiological findings were compared with postoperative CT scans by multiple reviewers blinded to patient identity or intraoperative findings. RESULTS Data were collected from 71 patients, in whom 802 screws were implanted between the T-1 and L-1 vertebral levels. A total of 32 screws ended up with screw threads encroaching on the spinal canal by at least 2 mm. Pulse-train stimulation within the pedicle track using a ball-tipped probe and electromyography from lower limb muscles correctly predicted all 32 (100%) of these medially malpositioned screws. The combination of pedicle track stimulation and electromyogram response from leg muscles proved to be far more effective in predicting these medially malpositioned screws than was direct screw stimulation and any of the target muscles (intercostal, abdominal, or lower limb muscles) we monitored. Based on receiver operating characteristic analysis, the combination of 10-mA (lower alarm) and 15-mA stimulation intensities proved most effective for detection of pedicle tracks that ultimately gave rise to medially malpositioned screws. Additional results pertaining to the impact of feedback of these test results on surgical decision making are provided in the companion report. CONCLUSIONS This novel neuromonitoring approach accurately predicts medially malpositioned thoracic screws. The approach could be readily implemented within any surgical program that is already using contemporary neuromonitoring methods that include transcranial stimulation for monitoring motor evoked potentials.

Tumor Fas (APO-1/CD95) up-regulation results in increased apoptosis and survival times for rats with intracranial malignant gliomas.

OBJECTIVEThe cellular “death” receptor Fas has been proposed to be a potential specific target for anti-glioma therapy. However, little is known regarding the effects of Fas expression on glioma viability in vivo. The goal of this study was to clarify the relationships among Fas expression, apoptosis, and survival rates for high-grade astrocytomas. METHODSFas expression was measured in several human glioblastoma multiforme cell lines and a malignant rat glioma cell line (36B10), before and after Fas up-regulation by gene transfer. Expression was correlated with the degree of Fas-mediated cytotoxicity and apoptosis induced after Fas activation. Subsequently, rats underwent intracranial implantation of either wild-type or genetically altered 36B10 cell lines, for study of the effects of Fas up-regulation on survival rates. RESULTSLow levels of cell surface Fas expression in glioblastomas multiforme were correlated with their limited susceptibility to Fas-mediated cytotoxicity. Through Fas receptor up-regulation, relationships among increased Fas expression, Fas-mediated cytotoxicity, and apoptosis were demonstrated. The percentage of cells undergoing apoptosis after exposure to a Fas ligand-producing cell line increased from 4% in the sham-transfected line (36B10−) to 27% in the Fas-transfected line (36B10-Fas). After intracranial implantation of these tumors into rats, the median survival time increased significantly from 14 days (36B10 and 36B10−) to 24.5 days (36B10-Fas), which represents a 75% increase in the survival time for the greater Fas-expressing group (P = 0.0005). CONCLUSIONIt seems that the overall low rate of apoptosis in high-grade astrocytomas is related to low levels of cell surface Fas expression. With increases in cellular Fas expression, rates of Fas-mediated apoptosis and survival rates were increased.

Impact of the number of metastatic brain lesions on survival after Gamma Knife radiosurgery

Effectiveness of Gamma Knife radiosurgery (GKRS: Elekta AB, Stockholm, Sweden) for patients with metastatic brain disease and the prognostic factors influencing their survival were analyzed in a 5 year retrospective data analysis (July 2001 to June 2006). Kaplan-Meier survival curves were constructed using univariate and multivariate analyses with the respective salient prognostic factors. This study analyzed data on 330 patients with brain metastases who underwent GKRS. Lung carcinoma (55%) was the most common primary cancer followed by breast (17.8%), melanoma (9.4%), colorectal (4.8%) and renal (3.9%). The median survival for all patients was 8 months. Survival ranged from 13 months for breast metastases, 10 months for renal, and 8 months for lung to 5 months for colorectal and melanoma. Mean age of patients was 58.5 years (range 18-81). Melanoma patients were younger with a mean age of 49 and also had the highest number of lesions (3.8) when compared to patients with renal (2.5), lung (2.8), colorectal (3) and breast (3.6). When stratified according to the number of lesions patient survival was 8 months (one to three lesions), 7.5 months (four or five lesions) and 7 months (six lesions or more). Mean Karnofsky Performance Status score (KPS) was 77 and survival dropped significantly from 8 months to 4.5 months if KPS was less than 70. Survival improved with a KPS of 70 or more, regardless of the number of lesions treated. Selection of patients based on the number of lesions may not be justified. A prospective trial is required to further define the prognostic factors affecting survival.