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Vaskuläre Anomalien (VA) manifestieren sich häufig im Kopf-Hals-Bereich und können zu lebensbedrohlichen Komplikationen führen. Obwohl in den letzten Jahren vereinzelte auslösende Genmutationen gefunden wurden, ist die genaue Entstehung der meisten vaskulären Anomalien noch nicht abschließend geklärt. In dieser Arbeit wurde die mögliche Rolle von humanen Papilloma-Viren (HPV) mittels immunhistochemischen Färbungen untersucht. Hierfür wurden insgesamt 27 VA-Proben untersucht und mikroskopisch beurteilt. Zur Kontrolle wurden dieselben immunhistochemischen Untersuchungen an 20 Oropharynxtumorproben durchgeführt. Es zeigte sich in 7 VA-Präparaten eine positive Reaktion des Gefäßendothels auf den Antikörper HPV16 E6/18 E6. Weiter konnte in 11 VA-Präparaten eine positive Reaktion von Nerven auf den gleichen Antikörper beobachtet werden. Die Untersuchungen mit den Antikörpern HPV16 E7, HPV16 E1/E4 und HPV18 E7 zeigten keine positiven Reaktionen. Die Untersuchung mit dem Anti-Human-Papillomavirus-Antikörper von Dako zeigte nur in drei Proben schwach positive Reaktionen von den insgesamt 27 untersuchten Proben. Die Färbungen mit dem anti-HPV16 L1-Antikörper (CAMVIR-1) zeigte bei allen Proben eine positive Reaktion in Muskeln, Epithelien der Haut, Endothelzellen und Nerven. Es zeigte sich jedoch in nur einem Präparat eine positive p16INK4a-Färbung der Gefäße, welche auch Ki67 negativ war, wodurch eine virale Ursache der p16INK4a-Hochregulation unwahrscheinlich erscheint. Die immunhistochemischen Färbungen ergaben zum großen Teil negative Ergebnisse. Nach ausführlicher Analyse sind die positiven Reaktionen des HPV16 L1 Antiköpers der Firma Santa Cruz Biotechnology, Inc. als unspezifische Reaktion zu werten. Ebenso deuten die Reaktionen des Antikörpers gegen HPV16 E6/18 E6 auf eine unspezifische Reaktion möglicherweise gegen β-Galaktosidase hin, da bei der Antikörperherstellung ein β-Galaktosidase-E6-Fusionsprotein eingesetzt wurde. In der vorliegenden rein explorativen Studie fand sich kein Anhaltspunkt dafür, dass in vaskulären Anomalien eine aktive virale Infektion vorliegt. Eine transiente Infektion, z.B. während der Schwangerschaft, die zu einer Veränderung der Gefäßbildung und zur Entstehung von vaskulären Anomalien führt, kann jedoch nicht ausgeschlossen werden. Dies sollte in weiteren Studien untersucht werden.

Additional file 4: The $$ \delta _{V}$$ δ V values of Volunteer 5 for the uncorrected Vw-maps as well as for the normalized Vw-maps using the diaphragm-based and the best and worst ROI-based normalization. The corresponding maps to these values are shown in Fig. 6.

Additional file 3: Boxplot comparison of ROI size and location for the Vw-maps. The boxplot for the signed mean deviations for each ROI using the $$ 8\times 8$$ 8 × 8 pixels square in (A) and the $$ 12\times 12$$ 12 × 12 pixels square in (B) compared to the uncorrected scans. Here, all scans of all volunteers were combined regardless of the slice position. The whiskers indicate 1.5 times the interquartile range (IQR). Outliers are not shown.

Additional file 2: Explanatory figure for the diaphragm-based normalization. The mean maxima and minima of the relative diaphragm positions $$ \overline{x}_{\text {ref,max}}$$ x ¯ ref,max and $$ \overline{x}_{\text {ref,min}}$$ x ¯ ref,min are extracted from the corresponding reference scan. The filtered average lung ventilation signal of scan i, which should be normalized, is determined and the mean maxima and minima of this signal ( $$ \overline{S}_{i,\text {max}}$$ S ¯ i , max , $$ \overline{S}_{i,\text {min}}$$ S ¯ i , min ) calculated from the respective peaks. This filtered average lung ventilation signal is also correlated with the relative diaphragm positions of scan i. Fitting this correlation allows to extract the slope $$ \text {d}S_{i}/ \text {d}x_{i}$$ d S i / d x i .

Additional file 1: The linear diaphragm position and lung signal correlation. The correlation between the relative diaphragm position and the average lung ventilation signal of each frame are exemplarily shown for a normal breathing scan of Volunteer 4 for aorta (A) and lung slice (B), deep breathing scan of Volunteer 2 (aorta (C) and lung (D)) as well as the shallow breathing scans of Volunteer 8 (aorta (E), lung (F)). The slope of the linear fit function depicted in red finds application in Eq. 1.

Purpose: Ventilation-induced tumour motion remains a challenge for the accuracy of proton therapy treatments in lung patients. We investigated the feasibility of using a 4D virtual CT (4D-vCT) approach based on deformable image registration (DIR) and motion-aware 4D CBCT reconstruction (MA-ROOSTER) to enable accurate daily proton dose calculation using a gantry-mounted CBCT scanner tailored to proton therapy. Methods: Ventilation correlated data of 10 breathing phases were acquired from a porcine ex-vivo functional lung phantom using CT and CBCT. 4D-vCTs were generated by (1) DIR of the mid-position 4D-CT to the mid-position 4D-CBCT (reconstructed with the MA-ROOSTER) using a diffeomorphic Morphons algorithm and (2) subsequent propagation of the obtained mid-position vCT to the individual 4D-CBCT phases. Proton therapy treatment planning was performed to evaluate dose calculation accuracy of the 4D-vCTs. A robust treatment plan delivering a nominal dose of 60 Gy was generated on the average intensity image of the 4D-CT for an approximated internal target volume (ITV). Dose distributions were then recalculated on individual phases of the 4D-CT and the 4D-vCT based on the optimized plan. Dose accumulation was performed for 4D-vCT and 4D-CT using DIR of each phase to the mid position, which was chosen as reference. Dose based on the 4D-vCT was then evaluated against the dose calculated on 4D-CT both, phase-by-phase as well as accumulated, by comparing dose volume histogram (DVH) values (Dmean, D2%, D98%, D95%) for the ITV, and by a 3D-gamma index analysis (global, 3%/3 mm, 5 Gy, 20 Gy and 30 Gy dose thresholds). Results: Good agreement was found between the 4D-CT and 4D-vCT-based ITV-DVH curves. The relative differences ((CT-vCT)/CT) between accumulated values of ITV Dmean, D2%, D95% and D98% for the 4D-CT and 4D-vCT-based dose distributions were −0.2%, 0.0%, −0.1% and −0.1%, respectively. Phase specific values varied between −0.5% and 0.2%, −0.2% and 0.5%, −3.5% and 1.5%, and −5.7% and 2.3%. The relative difference of accumulated Dmean over the lungs was 2.3% and Dmean for the phases varied between −5.4% and 5.8%. The gamma pass-rates with 5 Gy, 20 Gy and 30 Gy thresholds for the accumulated doses were 96.7%, 99.6% and 99.9%, respectively. Phase-by-phase comparison yielded pass-rates between 86% and 97%, 88% and 98%, and 94% and 100%. Conclusions: Feasibility of the suggested 4D-vCT workflow using proton therapy specific imaging equipment was shown. Results indicate the potential of the method to be applied for daily 4D proton dose estimation.

Additional file 1 Supplementary material.