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To explore the effect of sampling interval of training data acquisition on the intrafractional prediction error of surrogate signal-based dynamic tumor-tracking using a gimbal-mounted linac.Twenty pairs of respiratory motions were acquired from 20 patients (ten lung, five liver, and five pancreatic cancer patients) who underwent dynamic tumor-tracking with the Vero4DRT. First, respiratory motions were acquired as training data for an initial construction of the prediction model before the irradiation. Next, additional respiratory motions were acquired for an update of the prediction model due to the change of the respiratory pattern during the irradiation. The time elapsed prior to the second acquisition of the respiratory motion was 12.6 ± 3.1 min. A four-axis moving phantom reproduced patients' three dimensional (3D) target motions and one dimensional surrogate motions. To predict the future internal target motion from the external surrogate motion, prediction models were constructed by minimizing residual prediction errors for training data acquired at 80 and 320 ms sampling intervals for 20 s, and at 500, 1,000, and 2,000 ms sampling intervals for 60 s using orthogonal kV x-ray imaging systems. The accuracies of prediction models trained with various sampling intervals were estimated based on training data with each sampling interval during the training process. The intrafractional prediction errors for various prediction models were then calculated on intrafractional monitoring images taken for 30 s at the constant sampling interval of a 500 ms fairly to evaluate the prediction accuracy for the same motion pattern. In addition, the first respiratory motion was used for the training and the second respiratory motion was used for the evaluation of the intrafractional prediction errors for the changed respiratory motion to evaluate the robustness of the prediction models.The training error of the prediction model was 1.7 ± 0.7 mm in 3D for all sampling intervals. The intrafractional prediction error for the same motion pattern was 1.9 ± 0.7 mm in 3D for an 80 ms sampling interval, which increased larger than 1 mm in 10.0% of prediction models trained at a 2,000 ms sampling interval with a significant difference (P < 0.01) and up to 2.5% for the other sampling intervals without a significant difference (P > 0.05). The intrafractional prediction error for the changed respiratory motion pattern increased to 5.1 ± 2.4 mm in 3D for an 80 ms sampling interval; however, there was not a significant difference in the robustness of the prediction model between the 80 ms sampling interval and other sampling intervals (P > 0.05).Although the training error of the prediction model was consistent for the all sampling intervals, the prediction model using the larger sampling interval of the 2,000 ms increased the intrafractional prediction error for the same motion pattern. The realistic accuracy of the prediction model was difficult to estimate using the larger sampling interval during the training process. It is recommended to construct the prediction model at sampling interval ≤ 1,000 ms and to reconstruct the model during treatment.