With almost 900.000 new patients per year suffering from head and neck squamous cell carcinomas (HNSCC), who after radiotherapy treatments experience severe side effects, the focus is drawn to proton therapy. Proton therapy results to less side effects since healthy tissues surrounding the tumour receive less radiation dose. This results from the proton’s specific depth-dose profile with its Bragg peak.
To further reduce the effect on surrounding tissues, major improvements should be made in understanding the biologic response to proton therapy which will eventually lead to enlarging the therapeutic window. Here in vitro analysis with DNA damage repair inhibition or increasing proton dose rates can be performed to assess which DNA repair pathways repair radiation induced DNA damage and to assess the effect of proton dose rates on biological tissues. This will be investigated in vitro, while in vivo experiments are necessary as well. However, currently most mice are irradiated with protons in the plateau of the Bragg curve instead of the Bragg peak. Therefore, a dosimetric pipeline should be designed to allow for accurate placement of mouse tumours in the proton’s Bragg peak.
In this thesis, the osteosarcoma- and HNSCC-derived U2OS and FaDu cell lines were incubated with non-homologous end-joining or homologous recombination inhibitors during x-ray and proton irradiations. We have shown a radiosensitising effect of the non-homologous end joining inhibitor AZD7648 on FaDu cells during x-ray irradiation, but an effect during proton irradiation could not be proved nor neglected. The homologous recombination inhibitor B02 could not effectively inhibit homologous recombination and was shown to not affect clonogenic survival of FaDu during X-ray irradiations. The observed radiosensitising effect of the non-homologous recombination inhibitor AZD7648 can be exploited in patient selection based on already existing DNA damage repair deficiencies in the tumour. Furthermore, combination therapies could be used of photon irradiations with AZD7648 targeted to the tumour to artificially enlarge the therapeutic window.
Besides this, the HNSCC-derived FaDu cells were irradiated with varying dose rates to assess induction of a FLASH effect. This FLASH effect is usually observed after irradiations of >40 Gy/s resulting in a reduction of normal tissue complications while tumour control is maintained. In our analysis, small differences in low dose rates did not have any impact. The experimental set up for proton FLASH irradiations of cells was prepared and though we performed the first set of experiments no definite conclusion could be drawn due to the level of biological variation we observed.
Of course, differential effects between tumours and tissues like the FLASH effect should be investigated in vivo. Therefore, a set-up should be created to irradiate the mural tumour with Bragg peak protons. To reach this, two micro-CT scanners were calibrated to link Hounsfield units to stopping power ratios. The proton range in mice was determined and a 3D-printed mouse-like phantom was irradiated. In this thesis, determined stopping powers of all CIRS phantom inserts except for lung tissues were accurate with a maximum deviation of 2% or 6.5% after calibration with QuantumGx or VECTor micro-CT scanners. Larger deviations were observed for CIRS lung inserts or Gammex inserts. The irradiated gafchromic films inserted in the murinemorphic phantom showed dose distributions visualising the mouse’s anatomy, yet the dose was too low due to irradiation in the distal edge of the Bragg peak. The latter was confirmed with Monte Carlo simulations. This dosimetric set-up to place mice tumours in the proton Bragg peak should thus be slightly improved and can then be of great value for in vivo experiments of proton therapy. This enables execution of many new experiments with DNA damage repair inhibition and FLASH irradiations, potentially leading to an increase of the therapeutic window in proton therapy and less side effects for HNSCC-patients.

The goal of this research was to perform a 3D end-to-end test on a MR-linac to check the whole workflow using a clinical treatment plan. Dosimetric gel was used to obtain 3D spatial information, with the phantom in the same position for irradiation and scanning. In order to achieve this, fundamental elements of gel dosimetry needed to be investigated. In the MR-linac, irradiation is delivered in the presence of a permanent magnetic field. Therefore, the dosimetric response within a 1.5 T magnetic field should be validated. It is also important to investigate the time-dependence of the gel. It is preferable to read-out the gels within approximately one hour, so that the phantom does not have to be moved. Ideally, scanning and irradiation would be done at the same time, to see the dynamical dose delivery. The VIPAR gel was used for this research. The experiments demonstrated that R2 values for doses irradiated with magnetic field were the same as R2 values for the same dose irradiated without magnetic field. R2 values are still proportional to the dose. It was also shown that it is possible to scan the phantom within 20 minutes after irradiation. Sensitivity is at its highest after approximately 8 hours and stays stable afterwards, so scanning after 8 hours will improve the read-out accuracy. It was also possible to make a fit for the R2 versus time plots, which makes it possible to correct for change over time. The fit can be divided in two linear parts if time is plotted on a logarithmic scale, one fit for the time points before 7 hours, one for the time points after 7 hours. The partial doses acquired by the gel during radiation delivery were estimated. The equivalent R2 values then agreed with the extrapolated fit to within 4%. This is a good indication that dynamic gel (4D) dosimetry may be achievable. A protocol for a relative end-to-end test was also developed. From the preliminary results, it appeared that a relative end-to-end test can be performed with the read-out of gel within 1 hour. A new MR sequence needs to be developed. For this end-to-end test, the sequence needs to scan a larger volume with a higher resolution, therefore, the scan time will increase and real-time dosimetry will not be possible. Changing the MR sequence might also change the optimal irradiation-scanning interval and the R2 versus time curve. To perform absolute dosimetry, an extra calibration would be required.

Pencil beam scanning is becoming a more common treatment modality. However, its ability to deal with moving targets is known to be limited, as beam motion and target motion can reinforce each other, deteriorating the planned dose distribution in what is called the interplay effect. Literature concerning breathing motion usually investigates regular patterns. This work aims to investigate the magnitude of the interplay effect when considering irregular breathing signals. In silico calculations of dose distributions were made in the treatment planning system RayStation (version 7.99), using an XCAT phantom with 50 CT phases to model moving patient anatomy. An interplay calculator was included in RayStation, allowing calculation of disturbed doses based on a treatment plan and an irradiation time model for a proton therapy accelerator. The target investigated was a spherical liver tumour with 5cm diameter, irradiated with two beams delivering a prescription dose of 63 Gy. Plans without and with 5x layered repainting were created. Clinically realistic regular breathing patterns were generated to establish a baseline, after which irregularities were introduced. The basic form for all patterns was a sin^4 signal, with regular signal amplitudes ranging from 6 to 18 mm, period ranging from 3 to 4 s and phase between 0 and 2π rad. Considered irregularities were baseline shifts up to 34 mm, changing amplitudes between 6 and 18 mm, changing periods between 1.6 and 5.2 s and combinations. Evaluation was done by looking at dose homogeneity HI_5 and the fraction of the CTV volume that received a dose outside of the clinical limits of 95% and 107%, V_107/95. For the regular patterns, both a systematic and a randomised analysis were carried out. For irregular patterns, only a systematic analysis was carried out. The mean HI_5 was found to be 31% for regular patterns; the means of all irregular patterns stay below this, even though the size of the irregularities for some breathing patterns was very large. The mean V_107/95 was found to be 0.7 for regular patterns. Irregularities did not cause further deterioration. Five times layered repainting causes a statistically significant decrease in magnitude of the interplay effect across all breathing patterns by 50-80%, but is approximately 50% less effective against baseline shift than against other types of breathing. Interplay effect size correlates strongly with amplitude, but this correlation can be obscured because period and phase introduce very large variance. The interplay effect in general is large for investigated target size, prescription dose, beam configuration and machine performance. It can cause up to 100% of the CTV to receive a clinically unacceptable dose and lead to large inhomogeneities. Irregular breathing was not found to be notably worse. Repainting is effective, even against irregular breathing, but baseline shifts can undermine its effectiveness. Separately considering breathing irregularities for tumours similar to that investigated here is deemed of low importance; it is more important to properly model the magnitude of the interplay effect using an accurate, individualised breathing pattern.