GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Quantification of plan robustness against different uncertainty sources for classical and anatomical robust optimized treatment plans in head and neck cancer proton therapy.

OBJECTIVE: Classical robust optimization (cRO) in intensity-modulated proton therapy (IMPT) considers isocenter position and particle range uncertainties; anatomical robust optimization (aRO) aims to consider additional non-rigid positioning variations. This work compares the influence of different uncertainty sources on the robustness of cRO and aRO IMPT plans for head and neck squamous cell carcinoma (HNSCC). METHODS: Two IMPT plans were optimized for 20 HNSCC patients who received weekly control CTs (cCT): cRO, using solely the planning CT, and aRO, including 2 additional cCTs. The robustness of the plans in terms of clinical target volume (CTV) coverage and organ at risk (OAR) sparing was analyzed considering stepwise the influence of (1) non-rigid anatomical variations given by the weekly cCT, (2) with fraction-wise added rigid random setup errors and (3) additional systematic proton range uncertainties. RESULTS: cRO plans presented significantly higher nominal CTV coverage but are outperformed by aRO plans when considering non-rigid anatomical variations only, as cRO and aRO plans presented a median target coverage (D98%) decrease for the low-risk/high-risk CTV of 1.8/1.1 percentage points (pp) and -0.2 pp/-0.3 pp, respectively. Setup and range uncertainties had larger influence on cRO CTV coverage, but led to similar OAR dose changes in both plans. Considering all error sources, 10/2 cRO/aRO patients missed the CTV coverage and a limited number exceeded some OAR constraints in both plans. CONCLUSION: Non-rigid anatomical variations are mainly responsible for critical target coverage loss of cRO plans, whereas the aRO approach was robust against such variations. Both plans provide similar robustness of OAR parameters. ADVANCES IN KNOWLEDGE: The influence of different uncertainty sources was quantified for robust IMPT HNSCC plans.

Residual gammaH2AX foci in head and neck squamous cell carcinomas as predictors for tumour radiosensitivity: Evaluation in pre-clinical xenograft models and clinical specimens.

BACKGROUND AND PURPOSE: Predictive biomarkers can be instrumental to treatment individualisation of cancer patients and improve therapy outcome. Residual γH2AX foci represent a promising biomarker to predict tumour radiosensitivity. In this pre-clinical study, the slope of the dose-response curve was evaluated for its predictive relevance in head and neck squamous cell carcinoma xenografts (HNSCC). Additionally, the feasibility of the translated assay was tested in a clinical setting in patient derived HNSCC samples, and associations between residual γH2AX foci and clinical parameters were analysed. MATERIALS AND METHODS: Seven HNSCC xenografts models (FaDu, SAS, SKX, UT-SCC-5, UT-SCC-14, UT-SCC-45, XF354) were used. Tumour bearing NMRI nude mice were randomly distributed to five treatment arms (0-8 Gy). Residual γH2AX foci (24 h post irradiation) were counted by visual scoring in a micromilieu dependent manner (assessed with BrdU and pimonidazole). The local tumour control values measured as TCD50 (tumour control dose 50%) have previously been published. Patient derived HNSCC biopsies were cultivated ex vivo for 24 h including 4 h of pimonidazole and BrdU treatment, subsequently irradiated with 0-8 Gy and fixed after 24 h. RESULTS: In the pre-clinical study, the dose-response curve slopes negatively correlated with the tumour control dose after fractionated irradiation (TCD50,fx, R2 = 0.63, p = 0.032) and after single dose irradiation under homogeneous hypoxia (TCD50,SD,clamp, R2 = 0.66, p = 0.027). The γH2AX assay in clinical HNSCC samples showed a dose-response relationship, with the values of the slopes ranging from 0.099 Gy-1 to 0.920 Gy-1 (coefficient of variation = 52.8%). Slopes derived from patients were in the same ranges as the sensitive, moderate and resistant models of the pre-clinical study. Statistical analysis revealed a significant negative correlation between the slope and the patients' age (R2 = 0.65, p = 0.001). CONCLUSION: These results further support the promise of the slope of the residual γH2AX foci dose-response as a biomarker for radiosensitivity. In the clinical samples, the variation in the slopes reveals patients' specific repair capacities, which could hold potential value for treatment individualisation.

Utility of fiducial markers for target positioning in proton radiotherapy of oesophageal carcinoma.

BACKGROUND AND PURPOSE: Oesophageal mobility relative to bony anatomy is a major source of geometrical uncertainty in proton radiotherapy of oesophageal carcinoma. To mitigate this uncertainty we investigated the use of implanted fiducial markers for direct target verification in terms of safety, visibility, and stability. MATERIALS AND METHODS: A total of 19 helical gold markers were endoscopically implanted in ten patients. Their placement at the proximal and distal tumour borders was compared to tumour demarcations derived from [18F]Fluorodeoxyglucose positron emission tomography, their visibility quantified via the contrast-to-noise ratio on daily orthogonal X-ray imaging, and their mobility relative to bony anatomy analysed by means of retrospective triangulation. RESULTS: Marker implantation proceeded without complications, but the distal tumour border could not be reached in two patients. Marker locations corresponded reasonably well with metabolic tumour edges (mean: 5.4 mm more distally). Marker visibility was limited but mostly sufficient (mean contrast-to-noise ratio: 1.5), and sixteen markers (84%) remained in situ until the end of treatment. Overall, marker excursions from their planned position were larger than 5(10) mm in 59(17)% of all analysed fractions. On one occasion severe target displacement was only identified via markers and was corrected before treatment delivery. CONCLUSION: Implanted helical gold fiducial markers are a safe and reliable method of providing target-centric positioning verification in proton beam therapy of oesophageal carcinoma.

Including anatomical variations in robust optimization for head and neck proton therapy can reduce the need of adaptation.

BACKGROUND AND PURPOSE: Classical robust optimization considers uncertainties in patient setup and particle range. However, anatomical changes occurring during the treatment are neglected. Our aim was to compare classical robust optimization (cRO) with anatomical robust optimization (aRO), to quantify the influence of anatomical variations during the treatment course, and to assess the need of adaptation. MATERIALS AND METHODS: Planning CT and weekly control CTs (cCTs) from 20 head and neck patients were analysed. Three intensity-modulated proton therapy (IMPT) plans were compared: conventional PTV-based plan; cRO, using solely the planning CT, and aRO, including additionally the first 2 cCTs in the optimization. Weekly and total cumulative doses, considering anatomical variations during the treatment, were calculated and compared with the nominal plans. RESULTS: Nominal plans fulfilled clinical specifications for target coverage (D98% ≥95% of prescribed dose). The PTV-based and cRO approaches were not sufficient to account for anatomical changes during the treatment in 10 and 5 patients, respectively, resulting in the need of plan adaptation. With the aRO approach, in all except one patient the target coverage was conserved, and no adaptations were necessary. CONCLUSION: In 25% of the investigated cases, classical robust optimization is not sufficient to account for anatomical changes during the treatment. Adding additional information of random anatomical variations in the optimization improves plan robustness.

The clinical target volume in lung, head-and-neck, and esophageal cancer: Lessons from pathological measurement and recurrence analysis.

Radiotherapy research has achieved remarkable progress in target volume definition. Advances in medical imaging facilitate more precise localization of the gross tumor volume, alongside a more detailed understanding of the geometric uncertainties associated with treatment delivery that has enabled robust safety margins to be customized to the specific treatment scenario at hand. By contrast, the clinical target volume, meant to encompass gross tumor, as well as, adjacent sub-clinical disease, has evolved very little. It is more often defined by clinician experience and institutional convention than on a patient-specific basis. This disparity arises from the inherent invisibility of sub-clinical disease in current medical imaging. Its incidence and expanse can only be ascertained via indirect means. This article reviews two such strategies: histopathological measurements on resection specimen and analyses of locoregional recurrences after radiotherapy.

Aortic Arch Mapping by Computed Tomography for Actual Anatomic Studies in Times of Emerging Endovascular Therapies.

BACKGROUND: The latest advances in treatment of aortic arch pathologies increasingly included endovascular technologies. For those purposes, more detailed knowledge about the specific anatomic features are of particular interest, especially with regard on the need for better suitable stent grafts or even development of "off-the-shelf" stents. METHODS: The study enrolled patients undergoing computed tomography of the chest for other reasons than screening for aortic disease. Patients with aortic pathologies were excluded. Finally, 118 patients were included. Anatomic features of the aortic arch, the supra-aortic branches, distances and takeoff angles as well as specific diameters were assessed and analyzed with respect to the patients height, weight, age, and sex. RESULTS: A significant variability of all measurements was observed. Nonetheless, 4 recurrent types of aortic arch geometry were identified: (1) Classic arch (39%), (2) Gothic arch (39%), (3) Rectangle arch (11.9%), and (4) Plain arch (8.5%). Furthermore, the aortic diameter continuously decreased from the beginning of the ascending aorta during the after 200 mm by 24.8% (31.8 ± 3.6 mm to 23.9 ± 3.1 mm in mean). Distances from the aortic annulus to the supra-aortic branches takeoff points showed significant gender- and age-related differences with larger distances in the older and male (P < 0.001). Observed takeoff angles were 44.9 ± 15.9° for the brachiocephalic trunk, 25.7 ± 15.5° for the left common carotid artery, and 28.8 ± 14.5° for the left subclavian artery. CONCLUSIONS: Observed anatomic features were highly variable. It seemed that a "standard aortic arch" does not exist. Until today, the aortic arch as a dynamic and 3-dimensional entity is not fully understood. The development of "off-the-shelf" stents in the near future will be limited by this complexity and variability.