## 4 Discussions
[21] observed lower values in a range between \(-\)50.4% and 47.3% among PCa patients, and the authors concluded that this variation was due to the difficulty experienced by the patient in following the instructions for the correct rectal preparation. The patients in our study did not need to receive any preparation, since their lymph nodal metastases were treated, so that the percentage volume variations due to the different emptying statuses were even more pronounced (ranging from \(-\)99.2% up to 273.6%). Regarding the bladder volume variation, similar results were shown by [PERSON] et al. [21], reporting a variation range between \(-\)79.7% and 203.0%, in line with the findings of this study, with variations ranging from \(-\)74.2% up to 282.0%. In [PERSON] et al. [20], a lower mean displacement value for the bladder was shown regarding the lateral direction and anterior/posterior and inferior/superior directions, with displacements of 0.02 \(\pm\) 2.90, \(-\)0.86 \(\pm\) 6.24, and \(-\)3.40 \(\pm\) 8.80 mm, respectively. Both [PERSON] et al. [21] and [PERSON] et al. [23] considered patients with a full bladder and empty rectum. On the contrary, the differences reported in the present study are more marked, since the patients did not follow any instruction, as mentioned before. Regarding the cauda, the reference structure defined in order to validate our methodology was not affected by deformation and shrinkage, and a margin of only 3 mm was required to achieve the percentage volume of 97%, and the highest DSC value was observed, corroborating its static status. The discrepancies are mainly due to the contour shape and structure length variability between the s-CTs and CBCTs. Regarding the dosimetric impact of the volume variation, we observed a high degree of compliance with the constraints adopted by our institute. In general, a greater percentage dose variation was observed, with higher Mean_DA values and smaller DSC values (see Supplementary Materials: Figure S3). Reasonably, the closer the DSC index is to 1 (two volumes perfectly overlapped,) the smaller the Mean_DA value is, and the smaller the dose percentage variations are (clearly observed in the bladder and colon). The observed deviation did not show a predictable drift during the treatment course. As for the bladder and rectum, the volume variations are due to their expansion or contraction, caused by variations in their filling status [20,21]. Moreover, the rectum is more sensitive to variation in the shape of the anterior wall, as reported by [PERSON] et al. [35] for rectal cancer patients. The colon and ileum presented with pronounced daily shape variation due to deformation caused by mechanical stress, as highlighted by their low median DSC and high median Mean_DA within a very wide interval range (see Figure 3). Moreover, as emerged from the box plots, the rectum, ileum, and colon are more affected by outliers, a fact that may be linked to the random nature of their shape variation. Regarding the cauda, these effects are not evident, since it is the structure with the smallest range interval for both the DSC and Mean_DA (contouring variability). The main limitation of this study is related to the CBCT image quality. In fact, it is common knowledge from the literature that images produced by a fan beam CT (as CT OPTIMA) have a superior quality in terms of their clarity, uniformity, anatomical accuracy, and contrast resolution [33] compared to CBCT [36, 37, 38, 39, 40]. Therefore, the main difficulty was related to the fact that were dealing with nosier images and artefacts, which may have particularly undermined the delineation of the ileum and colon. The inter-observer variability was negligible, as only one physician was enrolled to contour all the OARs of interest. For this reason, the colon and ileum presented a lower median DSC with respect to the cauda.