A. Forbes

Changes in gene expression induced by chemoradiation in advanced cervical carcinoma : A microarray study of RTOG C-0128

PURPOSE To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting. METHODS RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Wards method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM). RESULTS From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA-IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n=5; squamous cell carcinoma, n=17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25. CONCLUSIONS Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.

SU‐DD‐A3‐03: Evaluation of An Infrared Camera and X‐Ray System for Gated Radiation Therapy

Purpose: To determine the clinical feasibility of a gated treatmentdelivery system from BrainLab™. Method and Materials: The Exactrac from BrainLab™ is a localization system that uses both infrared cameras and x‐rays. In gated mode, target location is determined by implanted fiducials. Breathing patterns are determined by infrared reflectors attached to the patients surface. The User selects an x‐ray trigger point and radiotherapy beam‐on window relative to the breathing cycle. Multiple trigger levels may be selected to simulate a fluoroscopic mode to measure organ motion. Prior to clinical use feasibility tests including localization accuracy, gating window accuracy, and beam‐on accuracy were performed. Patients with small lung lesions were selected for treatment and implanted with a 20 by 0.7 mm gold fiducial. Treatment planning CT scans were taken at expiration breath hold with internal and external fiducials present. Results:_Localization accuracy was within 3mm when using 20% of the breathing cycle for beam on. To date, five patients with lung lesions were treated.Treatment times were approximately twenty minutes (standard dose fractionations). Implanted fiducials were well localizable in all patients. Target motion was on the order of 5mm average. Repeat CT scans showed implants did not migrate. The primary limitations with the system were related to breathing signal due to placement of external fiducials. Conclusion: Gating treatment technique from Exactrac™ has been used to treatlung lesions. This initial evaluation of the system verified the accuracy of the localization system under Gated mode. Implanted fiducials are localizable in patients, and gating is possible. The benefit of this system is the potential to decrease treatment margins and improve targeting. Continued evaluation of this system would help to define patient specific dose margins and beam‐on windows for treatment.

TH‐C‐M100J‐03: Lung Localization and Initial Shifts Based On Implanted Markers

Purpose: The purpose of this study was to evaluate the daily setup error of small peripheral lungtumors based on implanted markers and stereoscopic x‐rays. Method and Materials:Twenty‐three patients with small peripheral lungtumors were implanted with radiographic markers for localized radiation therapy. Exhale CT scans were used for planning. At treatment, the patients were aligned to skin marks followed by exhale synchronized stereoscopic x‐rays (Exactrac™, BrainLAB). Setup error is evaluated as the shift from initial skin alignment to the implanted x‐ray alignment. The mean, σ mean, and RMS of σ for this data are reported. Results: A total of 790 daily treatment sessions on 23 patients were recorded. In all patients, an evaluation CT of the implanted marker was performed prior to treatment to verify marker location. The mean and σ for the 23 patients from initial setup to x‐ray localization were 0.2 ± 3.8 mm laterally, 1.6 ± 5.3 mm longitudinally, and −0.7 ± 7.8 mm vertically. The RMS error was Lat 5.5 mm, Long 7.7, and Vert 6.4 mm. Conclusion: A large patient study using implanted markers for lungtumor alignment indicated large initial setup shifts from skin marks. Daily random errors on the order of 8mm were reported. The range of these shifts is as large as 3.0 cm in some instances indicating that a small target would potentially be missed without better setup or with localization. The large systematic error is most likely due to the difference in tumor position from exhale breath hold (CT) relative to free breathing exhale (X‐Ray)..Conflict of Interest: This work is supported in part from a research grant from BrainLAB, Inc.