Sensitization of Patient-Derived Colorectal Cancer Organoids to Photon and Proton Radiation by Targeting DNA Damage Response Mechanisms.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient's tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

Orthotopic Glioblastoma Models for Evaluation of the Clinical Target Volume Concept.

In times of high-precision radiotherapy, the accurate and precise definition of the primary tumor localization and its microscopic spread is of enormous importance. In glioblastoma, the microscopic tumor extension is uncertain and, therefore, population-based margins for Clinical Target Volume (CTV) definition are clinically used, which could either be too small-leading to increased risk of loco-regional recurrences-or too large, thus, enhancing the probability of normal tissue toxicity. Therefore, the aim of this project is to investigate an individualized definition of the CTV in preclinical glioblastoma models based on specific biological tumor characteristics. The microscopic tumor extensions of two different orthotopic brain tumor models (U87MG_mCherry; G7_mCherry) were evaluated before and during fractionated radiotherapy and correlated with corresponding histological data. Representative tumor slices were analyzed using Matrix-Assisted Laser Desorption/Ionization (MALDI) and stained for putative stem-like cell markers as well as invasion markers. The edges of the tumor are clearly shown by the MALDI segmentation via unsupervised clustering of mass spectra and are consistent with the histologically defined border in H&E staining in both models. MALDI component analysis identified specific peaks as potential markers for normal brain tissue (e.g., 1339 m/z), whereas other peaks demarcated the tumors very well (e.g., 1562 m/z for U87MG_mCherry) irrespective of treatment. MMP14 staining revealed only a few positive cells, mainly in the tumor border, which could reflect the invasive front in both models. The results of this study indicate that MALDI information correlates with microscopic tumor spread in glioblastoma models. Therefore, an individualized CTV definition based on biological tumor characteristics seems possible, whereby the visualization of tumor volume and protein heterogeneity can be potentially used to define radiotherapy-sensitive and resistant areas.

Combined Systemic Drug Treatment with Proton Therapy: Investigations on Patient-Derived Organoids.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Impact of Blood Parameters and Normal Tissue Dose on Treatment Outcome in Esophageal Cancer Patients Undergoing Neoadjuvant Radiochemotherapy.

Despite technological advances, normal tissue sparing in photon beam irradiation is still challenging. Since in esophageal cancer this may inflict damage on the lungs, heart and bone marrow, possibly impacting on outcome, the aim of this study was to investigate the association of normal tissue dose and blood parameters on the survival of patients having undergone neoadjuvant radiochemotherapy (RCTx) followed by surgery. This retrospective study included 125 patients irradiated to 40−41.4 Gy with photons or protons combined with concurrent chemotherapy. On initial and restaging 18F-FDG-PET/CT, the lungs and heart were contoured as organs at risk for which standardized uptake values (SUV) were evaluated. The mean radiation dose (Dmean) to the lungs and heart, the volume of the lungs receiving at least 20 Gy (V20Gy_lung) and various pre- and per-treatment blood parameters were included in the Cox regression analyses. Results: The median follow-up time was 19.8 months and median overall survival 37 months (95% confidence interval: 16−58.9 months). In multivariate analysis, higher radiation doses to the lungs and heart were statistically significantly associated with decreased overall survival (Dmean_lung: p < 0.001; V20Gy_lung: p < 0.002; Dmean_heart: p = 0.005). Neither the 18F-FDG-PET nor blood parameters were predictive for overall survival. In patients with locally advanced esophageal cancer treated with RCTx, the radiation dose to the heart and lungs was significantly associated with overall survival.

Treatment planning comparison in the PROTECT-trial randomising proton versus photon beam therapy in oesophageal cancer: Results from eight European centres.

PURPOSE: To compare dose distributions and robustness in treatment plans from eight European centres in preparation for the European randomized phase-III PROTECT-trial investigating the effect of proton therapy (PT) versus photon therapy (XT) for oesophageal cancer. MATERIALS AND METHODS: All centres optimized one PT and one XT nominal plan using delineated 4DCT scans for four patients receiving 50.4 Gy (RBE) in 28 fractions. Target volume receiving 95% of prescribed dose (V95%iCTVtotal) should be >99%. Robustness towards setup, range, and respiration was evaluated. The plans were recalculated on a surveillance 4DCT (sCT) acquired at fraction ten and robustness evaluation was performed to evaluate the effect of respiration and inter-fractional anatomical changes. RESULTS: All PT and XT plans complied with V95%iCTVtotal >99% for the nominal plan and V95%iCTVtotal >97% for all respiratory and robustness scenarios. Lung and heart dose varied considerably between centres for both modalities. The difference in mean lung dose and mean heart dose between each pair of XT and PT plans was in median [range] 4.8 Gy [1.1;7.6] and 8.4 Gy [1.9;24.5], respectively. Patients B and C showed large inter-fractional anatomical changes on sCT. For patient B, the minimum V95%iCTVtotal in the worst-case robustness scenario was 45% and 94% for XT and PT, respectively. For patient C, the minimum V95%iCTVtotal was 57% and 72% for XT and PT, respectively. Patient A and D showed minor inter-fractional changes and the minimum V95%iCTVtotal was >85%. CONCLUSION: Large variability in dose to the lungs and heart was observed for both modalities. Inter-fractional anatomical changes led to larger target dose deterioration for XT than PT plans.

Radiomics-based tumor phenotype determination based on medical imaging and tumor microenvironment in a preclinical setting.

BACKGROUND AND PURPOSE: Radiomics analyses have been shown to predict clinical outcomes of radiotherapy based on medical imaging-derived biomarkers. However, the biological meaning attached to such image features often remains unclear, thus hindering the clinical translation of radiomics analysis. In this manuscript, we describe a preclinical radiomics trial, which attempts to establish correlations between the expression of histological tumor microenvironment (TME)- and magnetic resonance imaging (MRI)-derived image features. MATERIALS & METHODS: A total of 114 mice were transplanted with the radioresistant and radiosensitive head and neck squamous cell carcinoma cell lines SAS and UT-SCC-14, respectively. The models were irradiated with five fractions of protons or photons using different doses. Post-treatment T1-weighted MRI and histopathological evaluation of the TME was conducted to extract quantitative features pertaining to tissue hypoxia and vascularization. We performed radiomics analysis with leave-one-out cross validation to identify the features most strongly associated with the tumor's phenotype. Performance was assessed using the area under the curve (AUCValid) and F1-score. Furthermore, we analyzed correlations between TME- and MRI features using the Spearman correlation coefficient ρ. RESULTS: TME and MRI-derived features showed good performance (AUCValid,TME = 0.72, AUCValid,MRI = 0.85, AUCValid,Combined=0.85) individual tumor phenotype prediction. We found correlation coefficients of ρ=-0.46 between hypoxia-related TME features and texture-related MRI features. Tumor volume was a strong confounder for MRI feature expression. CONCLUSION: We demonstrated a preclinical radiomics implementation and notable correlations between MRI- and TME hypoxia-related features. Developing additional TME features may help to further unravel the underlying biology.

Value of PET imaging for radiation therapy.

This comprehensive review written by experts in their field gives an overview on the current status of incorporating positron emission tomography (PET) into radiation treatment planning. Moreover, it highlights ongoing studies for treatment individualisation and per-treatment tumour response monitoring for various primary tumours. Novel tracers and image analysis methods are discussed. The authors believe this contribution to be of crucial value for experts in the field as well as for policy makers deciding on the reimbursement of this powerful imaging modality.

Value of PET imaging for radiation therapy.

This comprehensive review written by experts in their field gives an overview on the current status of incorporating positron emission tomography (PET) into radiation treatment planning. Moreover, it highlights ongoing studies for treatment individualisation and per-treatment tumour response monitoring for various primary tumours. Novel tracers and image analysis methods are discussed. The authors believe this contribution to be of crucial value for experts in the field as well as for policy makers deciding on the reimbursement of this powerful imaging modality.

Identification of patient benefit from proton beam therapy in brain tumour patients based on dosimetric and NTCP analyses.

BACKGROUND: The limited availability of proton beam therapy (PBT) requires individual treatment selection strategies, such as the model-based approach. In this study, we assessed the dosimetric benefit of PBT compared to photon therapy (XRT), analysed the corresponding changes in normal tissue complication probability (NTCP) on a variety of available models, and illustrated model-based patient selection in an in-silico study for patients with brain tumours. METHODS: For 92 patients treated at two PBT centres, volumetric modulated arc therapy treatment plans were retrospectively created for comparison with the clinically applied PBT plans. Several dosimetric parameters for the brain excluding tumour and margins, cerebellum, brain stem, frontal and temporal lobes, hippocampi, cochleae, chiasm, optic nerves, lacrimal glands, lenses, pituitary gland, and skin were compared between both modalities using Wilcoxon signed-rank tests. NTCP differences (ΔNTCP) were calculated for 11 models predicting brain necrosis, delayed recall, temporal lobe injury, hearing loss, tinnitus, blindness, ocular toxicity, cataract, endocrine dysfunction, alopecia, and erythema. A patient was assumed to be selected for PBT if ΔNTCP exceeded a threshold of 10 percentage points for at least one of the side-effects. RESULTS: PBT substantially reduced the dose in almost all investigated OARs, especially in the low and intermediate dose ranges and for contralateral organs. In general, NTCP predictions were significantly lower for PBT compared to XRT, in particular in ipsilateral organs. Considering ΔNTCP of all models, 80 patients (87.0%) would have been selected for PBT in this in-silico study, mainly due to predictions of a model on delayed recall (51 patients). CONCLUSION: In this study, substantial dose reductions for PBT were observed, mainly in contralateral organs. However, due to the sigmoidal dose response, NTCP was particularly reduced in ipsilateral organs. This underlines that physical dose-volume parameters alone may not be sufficient to describe the clinical relevance between different treatment techniques and highlights potential benefits of NTCP models. Further NTCP models for different modern treatment techniques are mandatory and existing models have to be externally validated in order to implement the model-based approach in clinical practice for cranial radiotherapy.

Modelling of late side-effects following cranial proton beam therapy.

BACKGROUND: The limited availability of proton beam therapy (PBT) requires individual treatment selection strategies that can be based on normal tissue complication probability (NTCP) models. We developed and externally validated NTCP models for common late side-effects following PBT in brain tumour patients to optimise patients' quality of life. METHODS: Cohorts from three PBT centres (216 patients) were investigated for several physician-rated endpoints at 12 and 24 months after PBT: alopecia, dry eye syndrome, fatigue, headache, hearing and memory impairment, and optic neuropathy. Dose-volume parameters of associated normal tissues and clinical factors were used for logistic regression modelling in a development cohort. Statistically significant parameters showing high area under the receiver operating characteristic curve (AUC) values in internal cross-validation were externally validated. In addition, analyses of the pooled cohorts and of time-dependent generalised estimating equations including all patient data were performed. RESULTS: In the validation study, mild alopecia was related to high dose parameters to the skin [e.g. the dose to 2% of the volume (D2%)] at 12 and 24 months after PBT. Mild hearing impairment at 24 months after PBT was associated with the mean dose to the ipsilateral cochlea. Additionally, the pooled analyses revealed dose-response relations between memory impairment and intermediate to high doses to the remaining brain as well as D2% of the hippocampi. Mild fatigue at 24 months after PBT was associated with D2% to the brainstem as well as with concurrent chemotherapy. Moreover, in generalised estimating equations analysis, dry eye syndrome was associated with the mean dose to the ipsilateral lacrimal gland. CONCLUSION: We developed and in part validated NTCP models for several common late side-effects following PBT in brain tumour patients. Validation studies are required for further confirmation.

Proposal for the delineation of neoadjuvant target volumes in oesophageal cancer.

PURPOSE: To define instructions for delineation of target volumes in the neoadjuvant setting in oesophageal cancer. MATERIALS AND METHODS: Radiation oncologists of five European centres participated in the following consensus process: [1] revision of published (MEDLINE) and national/institutional delineation guidelines; [2] first delineation round of five cases (patient 1-5) according to national/institutional guidelines; [3] consensus meeting to discuss the results of step 1 and 2, followed by a target volume delineation proposal; [4] circulation of proposed instructions for target volume delineation and atlas for feedback; [5] second delineation round of five new cases (patient 6-10) to peer review and validate (two additional centres) the agreed delineation guidelines and atlas; [6] final consensus on the delineation guidelines depicted in an atlas. Target volumes of the delineation rounds were compared between centres by Dice similarity coefficient (DSC) and maximum/mean undirected Hausdorff distances (Hmax/Hmean). RESULTS: In the first delineation round, the consistency between centres was moderate (CTVtotal: DSC = 0.59-0.88; Hmean = 0.2-0.4 cm). Delineations in the second round were much more consistent. Lowest variability was obtained between centres participating in the consensus meeting (CTVtotal: DSC: p < 0.050 between rounds for patients 6/7/8/10; Hmean: p < 0.050 for patients 7/8/10), compared to validation centres (CTVtotal: DSC: p < 0.050 between validation and consensus meeting centres for patients 6/7/8; Hmean: p < 0.050 for patients 7/10). A proposal for delineation of target volumes and an atlas were generated. CONCLUSION: We proposed instructions for target volume delineation and an atlas for the neoadjuvant radiation treatment in oesophageal cancer. These will enable a more uniform delineation of patients in clinical practice and clinical trials.

A convolutional neural network for fully automated blood SUV determination to facilitate SUR computation in oncological FDG-PET.

PURPOSE: The standardized uptake value (SUV) is widely used for quantitative evaluation in oncological FDG-PET but has well-known shortcomings as a measure of the tumor's glucose consumption. The standard uptake ratio (SUR) of tumor SUV and arterial blood SUV (BSUV) possesses an increased prognostic value but requires image-based BSUV determination, typically in the aortic lumen. However, accurate manual ROI delineation requires care and imposes an additional workload, which makes the SUR approach less attractive for clinical routine. The goal of the present work was the development of a fully automated method for BSUV determination in whole-body PET/CT. METHODS: Automatic delineation of the aortic lumen was performed with a convolutional neural network (CNN), using the U-Net architecture. A total of 946 FDG PET/CT scans from several sites were used for network training (N = 366) and testing (N = 580). For all scans, the aortic lumen was manually delineated, avoiding areas affected by motion-induced attenuation artifacts or potential spillover from adjacent FDG-avid regions. Performance of the network was assessed using the fractional deviations of automatically and manually derived BSUVs in the test data. RESULTS: The trained U-Net yields BSUVs in close agreement with those obtained from manual delineation. Comparison of manually and automatically derived BSUVs shows excellent concordance: the mean relative BSUV difference was (mean ± SD) = (- 0.5 ± 2.2)% with a 95% confidence interval of [- 5.1,3.8]% and a total range of [- 10.0, 12.0]%. For four test cases, the derived ROIs were unusable (< 1 ml). CONCLUSION: CNNs are capable of performing robust automatic image-based BSUV determination. Integrating automatic BSUV derivation into PET data processing workflows will significantly facilitate SUR computation without increasing the workload in the clinical setting.

Establishment and Characterisation of Heterotopic Patient-Derived Xenografts for Glioblastoma.

Glioblastoma is an aggressive brain tumour with a patient median survival of approximately 14 months. The development of innovative treatment strategies to increase the life span and quality of life of patients is hence essential. This requires the use of appropriate glioblastoma models for preclinical testing, which faithfully reflect human cancers. The aim of this study was to establish glioblastoma patient-derived xenografts (PDXs) by heterotopic transplantation of tumour pieces in the axillae of NMRI nude mice. Ten out of 22 patients' samples gave rise to tumours in mice. Their human origin was confirmed by microsatellite analyses, though minor changes were observed. The glioblastoma nature of the PDXs was corroborated by pathological evaluation. Latency times spanned from 48.5 to 370.5 days in the first generation. Growth curve analyses revealed an increase in the growth rate with increasing passages. The methylation status of the MGMT promoter in the primary material was maintained in the PDXs. However, a trend towards a more methylated pattern could be found. A correlation was observed between the take in mice and the proportion of Sox2+ cells (r = 0.49, p = 0.016) and nestin+ cells (r = 0.55, p = 0.007). Our results show that many PDXs maintain key features of the patients' samples they derive from. They could thus be used as preclinical models to test new therapies and biomarkers.

Specific requirements for translation of biological research into clinical radiation oncology.

Radiotherapy has been optimized over the last decades not only through technological advances, but also through the translation of biological knowledge into clinical treatment schedules. Optimization of fractionation schedules and/or the introduction of simultaneous combined systemic treatment have significantly improved tumour cure rates in several cancer types. With modern techniques, we are currently able to measure factors of radiation resistance or radiation sensitivity in patient tumours; the definition of new biomarkers is expected to further enable personalized treatments. In this Review article, we overview important translation paths and summarize the quality requirements for preclinical and translational studies that will help to avoid bias in trial results.

High-precision image-guided proton irradiation of mouse brain sub-volumes.

BACKGROUND AND PURPOSE: Proton radiotherapy offers the potential to reduce normal tissue toxicity. However, clinical safety margins, range uncertainties, and varying relative biological effectiveness (RBE) may result in a critical dose in tumor-surrounding normal tissue. To assess potential adverse effects in preclinical studies, image-guided proton mouse brain irradiation and analysis of DNA damage repair was established. MATERIAL AND METHODS: We designed and characterized a setup to shape proton beams with 7 mm range in water and 3 mm in diameter and commissioned a Monte Carlo model for in vivo dose simulation. Cone-beam computed tomography and orthogonal X-ray imaging were used to delineate the right hippocampus and position the mice. The brains of three C3H/HeNRj mice were irradiated with 8 Gy and excised 30 min later. Initial DNA double-strand breaks were visualized by staining brain sections for cell nuclei and γH2AX. Imaged sections were analyzed with an automated and validated processing pipeline to provide a quantitative, spatially resolved radiation damage indicator. RESULTS: The analyzed DNA damage pattern clearly visualized the radiation effect in the mouse brains and could be mapped to the simulated dose distribution. The proton beam passed the right hippocampus and stopped in the central brain region for all evaluated mice. CONCLUSION: We established image-guided proton irradiation of mouse brains. The clinically oriented workflow facilitates (back-) translational studies. Geometric accuracy, detailed Monte Carlo dose simulations, and cell-based assessment enable a biologically and spatially resolved analysis of radiation response and RBE.

Combined tumor plus nontumor interim FDG-PET parameters are prognostic for response to chemoradiation in squamous cell esophageal cancer.

We have investigated the prognostic value of two novel interim 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) parameters in patients undergoing chemoradiation (CRT) for esophageal squamous cell carcinoma (ESCC): one tumor parameter (maximal standardized uptake ratio rSUR) and one normal tissue parameter (change of FDG uptake within irradiated nontumor-affected esophagus ∆SUVNTO ). PET data of 134 European and Chinese patients were analyzed. Parameter establishment was based on 36 patients undergoing preoperative CRT plus surgery, validation was performed in 98 patients receiving definitive CRT. Patients received PET imaging prior and during fourth week of CRT. Clinical parameters, baseline PET parameters, and interim PET parameters (rSUR and ∆SUVNTO ) were analyzed and compared to event-free survival (EFS), overall survival (OS), loco-regional control (LRC) and freedom from distant metastases (FFDM). Combining rSUR and ∆SUVNTO revealed a strong prognostic impact on EFS, OS, LRC and FFDM in patients undergoing preoperative CRT. In the definitive CRT cohort, univariate analysis with respect to EFS revealed several staging plus both previously established interim PET parameters as significant prognostic factors. Multivariate analyses revealed only rSUR and ∆SUVNTO as independent prognostic factors (p = 0.003, p = 0.008). Combination of these parameters with the cutoff established in preoperative CRT revealed excellent discrimination of patients with a long or short EFS (73% vs. 17% at 2 years, respectively) and significantly discriminated all other endpoints (OS, p < 0.001; LRC, p < 0.001; FFDM, p = 0.02), even in subgroups. Combined use of interim FDG-PET derived parameters ∆SUVNTO and rSUR seems to have predictive potential, allowing to select responders for definitive CRT and omission of surgery.

Neurocognitive function and quality of life after proton beam therapy for brain tumour patients.

BACKGROUND: Neurocognitive function of adult patients with brain tumours may deteriorate after radiotherapy. Proton beam therapy (PBT) reduces the volume of irradiated healthy brain tissue and could potentially preserve neurocognition and quality of life (QoL). As present data are still limited, the impact of clinical factors and dosimetric parameters on neurocognitive function and QoL during recurrence-free follow-up after PBT is investigated. METHODS: The current study includes 62 brain tumour patients treated with PBT between 2015 and 2017. Neurocognition and QoL were assessed at baseline and every 3 months after PBT using the Montreal Cognitive Assessment (MoCA) test together with EORTC-QLQ-C30 and BN20 questionnaires, respectively. Objective and self-reported measures of neurocognitive functions were correlated. During two years of follow-up, the impact of clinical co-factors as well as dosimetric parameters of several brain structures were analysed using a mixed-model approach. RESULTS: At baseline, mean MoCA total score was 24.8/30 and self-reported cognitive function was 68.9/100. Both remained stable over time. Patients with impaired neurocognition on the MoCA test reported significantly lower global health status, cognitive, physical and role function as well as more fatigue, pain, headache and communication deficits compared to normal performing patients. For most follow-up time points, the majority of MoCA subitems correlated significantly to QoL items regarding neurocognition. Slight deterioration of the MoCA score was associated with tumours located in the left hemisphere and with an increase in relative volume of the anterior cerebellum that received doses of 30-40 Gy(RBE). CONCLUSION: Self-reported and objectively measured neurocognition and most other QoL domains remained largely stable over time during recurrence-free follow-up for brain tumour patients treated with PBT. The association between reduced cognitive function and irradiated volume of the anterior cerebellum requires validation in larger studies and comparison to patients treated with photon therapy.