Daniela Trani

Enhanced intestinal tumor multiplicity and grade in vivo after HZE exposure: mouse models for space radiation risk estimates

Carcinogenesis induced by space radiation is considered a major risk factor in manned interplanetary and other extended missions. The models presently used to estimate the risk for cancer induction following deep space radiation exposure are based on data from A-bomb survivor cohorts and do not account for important biological differences existing between high-linear energy transfer (LET) and low-LET-induced DNA damage. High-energy and charge (HZE) radiation, the main component of galactic cosmic rays (GCR), causes highly complex DNA damage compared to low-LET radiation, which may lead to increased frequency of chromosomal rearrangements, and contribute to carcinogenic risk in astronauts. Gastrointestinal (GI) tumors are frequent in the United States, and colorectal cancer (CRC) is the third most common cancer accounting for 10% of all cancer deaths. On the basis of the aforementioned epidemiological observations and the frequency of spontaneous precancerous GI lesions in the general population, even a modest increase in incidence by space radiation exposure could have a significant effect on health risk estimates for future manned space flights. Ground-based research is necessary to reduce the uncertainties associated with projected cancer risk estimates and to gain insights into molecular mechanisms involved in space-induced carcinogenesis. We investigated in vivo differential effects of γ-rays and HZE ions on intestinal tumorigenesis using two different murine models, ApcMin/+ and Apc1638N/+. We showed that γ- and/or HZE exposure significantly enhances development and progression of intestinal tumors in a mutant-line-specific manner, and identified suitable models for in vivo studies of space radiation–induced intestinal tumorigenesis.

Accelerated hematopoietic toxicity by high energy (56)Fe radiation.

Purpose: There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or X-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. Methods: C57BL/6J mice were irradiated with 56Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of 56Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Results: Although onset was more rapid, 56Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy, respectively, with relative biologic effectiveness for 56Fe ions of 1.25 and 1.06 for protons. Conclusions: 56Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

High-Energy Particle-Induced Tumorigenesis Throughout the Gastrointestinal Tract

Epidemiological data reveals the gastrointestinal (GI) tract as one of the main sites for low-LET radiation-induced cancers. Importantly, the use of particle therapy is increasing, but cancer risk by high-LET particles is still poorly understood. This gap in our knowledge also remains a major limiting factor in planning long-term space missions. Therefore, assessing risks and identifying predisposing factors for carcinogenesis induced by particle radiation is crucial for both astronauts and cancer survivors. We have previously shown that exposure to relatively high doses of high-energy 56Fe ions induced higher intestinal tumor frequency and grade in the small intestine of ApcMin/+ mice than γ rays. However, due to the high number of spontaneous lesions (∼30) that develop in ApcMin/+ animals, this Apc mutant model is not suitable to investigate effects of cumulative doses <1 Gy, which are relevant for risk assessment in astronauts and particle radiotherapy patients. However, Apc1638N/+ mice develop a relatively small number of spontaneous lesions (∼3 per animal) in both small intestine and colon, and thus we propose a better model for studies on radiation-induced carcinogenesis. Here, we investigated model particle radiation increases tumor frequency and grade in the entire gastrointestinal tract (stomach and more distal intestine) after high- and low-radiation doses whether in the Apc1638N/+. We have previously reported that an increase in small intestinal tumor multiplicity after exposure to γ rays was dependent on gender in Apc1638N/+ mice, and here we investigated responses to particle radiation in the same model. Phenotypical and histopathological observations were accompanied by late changes in number and position of mitotic cells in intestinal crypts from animals exposed to different radiation types.

Preclinical assessment of efficacy of radiation dose painting based on intratumoral FDG-PET uptake

Purpose: We tested therapeutic efficacy of two dose painting strategies of applying higher radiation dose to tumor subvolumes with high FDG uptake (biologic target volume, BTV): dose escalation and dose redistribution. We also investigated whether tumor response was determined by the highest dose in BTV or the lowest dose in gross tumor volume (GTV). Experimental Design: FDG uptake was evaluated in rat rhabdomyosarcomas prior to irradiation. BTV was defined as 30% of GTV with the highest (BTVhot) or lowest (BTVcold) uptake. To test efficacy of dose escalation, tumor response (time to reach two times starting tumor volume, TGTV2) to Hot Boost irradiation (40% higher dose to BTVhot) was compared with Cold Boost (40% higher dose to BTVcold), while mean dose to GTV remained 12 Gy. To test efficacy of dose redistribution, TGTV2 after Hot Boost was compared with uniform irradiation with the same mean dose (8 or 12 Gy). Results: TGTV2 after 12 Gy delivered heterogeneously (Hot and Cold Boost) or uniformly were not significantly different: 20.2, 19.5, and 20.6 days, respectively. Dose redistribution (Hot Boost) with 8 Gy resulted in faster tumor regrowth as compared with uniform irradiation (13.3 vs. 17.1 days; P = 0.026). Further increase in dose gradient to 60% led to a more pronounced decrease in TGTV2 (10.9 days; P < 0.0001). Conclusions: Dose escalation effect was independent of FDG uptake in target tumor volume, while dose redistribution was detrimental in this tumor model for dose levels applied here. Our data are consistent with the hypothesis that tumor response depends on the minimum intratumoral dose. Clin Cancer Res; 21(24); 5511–8. ©2015 AACR.

Metabolomic Profiling of Urine Samples from Mice Exposed to Protons Reveals Radiation Quality and Dose Specific Differences

As space travel is expanding to include private tourism and travel beyond low-Earth orbit, so is the risk of exposure to space radiation. Galactic cosmic rays and solar particle events have the potential to expose space travelers to significant doses of radiation that can lead to increased cancer risk and other adverse health consequences. Metabolomics has the potential to assess an individuals risk by exploring the metabolic perturbations in a biofluid or tissue. In this study, C57BL/6 mice were exposed to 0.5 and 2 Gy of 1 GeV/nucleon of protons and the levels of metabolites were evaluated in urine at 4 h after radiation exposure through liquid chromatography coupled to time-of-flight mass spectrometry. Significant differences were identified in metabolites that map to the tricarboxylic acid (TCA) cycle and fatty acid metabolism, suggesting that energy metabolism is severely impacted after exposure to protons. Additionally, various pathways of amino acid metabolism (tryptophan, tyrosine, arginine and proline and phenylalanine) were affected with potential implications for DNA damage repair and cognitive impairment. Finally, presence of products of purine and pyrimidine metabolism points to direct DNA damage or increased apoptosis. Comparison of these metabolomic data to previously published data from our laboratory with gamma radiation strongly suggests a more pronounced effect on metabolism with protons. This is the first metabolomics study with space radiation in an easily accessible biofluid such as urine that further investigates and exemplifies the biological differences at early time points after exposure to different radiation qualities.

Sex-dependent differences in intestinal tumorigenesis induced in Apc1638N/+ mice by exposure to γ rays.

PURPOSE The purpose of the present study was to assess the effect of 1 and 5 Gy radiation doses and to investigate the interplay of gender and radiation with regard to intestinal tumorigenesis in an adenomatous polyposis coli (APC) mutant mouse model. METHODS AND MATERIALS Apc1638N/+ female and male mice were exposed whole body to either 1 Gy or 5 Gy of γ rays and euthanized when most of the treated mice became moribund. Small and large intestines were processed to determine tumor burden, distribution, and grade. Expression of proliferation marker Ki-67 and estrogen receptor (ER)-α were also assessed by immunohistochemistry. RESULTS We observed that, with both 1 Gy and 5 Gy of γ rays, females displayed reduced susceptibility to radiation-induced intestinal tumorigenesis compared with males. As for radiation effect on small intestinal tumor progression, although no substantial differences were found in the relative frequency and degree of dysplasia of adenomas in irradiated animals compared with controls, invasive carcinomas were found in 1-Gy- and 5-Gy-irradiated animals. Radiation exposure was also shown to induce an increase in protein levels of proliferation marker Ki-67 and sex-hormone receptor ER-α in both non tumor mucosa and intestinal tumors from irradiated male mice. CONCLUSIONS We observed important sex-dependent differences in susceptibility to radiation-induced intestinal tumorigenesis in Apc1638N/+ mutants. Furthermore, our data provide evidence that exposure to radiation doses as low as 1 Gy can induce a significant increase in intestinal tumor multiplicity as well as enhance tumor progression in vivo.

What level of accuracy is achievable for preclinical dose painting studies on a clinical irradiation platform

Advancements made over the past decades in both molecular imaging and radiotherapy planning and delivery have enabled studies that explore the efficacy of heterogeneous radiation treatment (“dose painting”) of solid cancers based on biological information provided by different imaging modalities. In addition to clinical trials, preclinical studies may help contribute to identifying promising dose painting strategies. The goal of this current study was twofold: to develop a reproducible positioning and set-up verification protocol for a rat tumor model to be imaged and treated on a clinical platform, and to assess the dosimetric accuracy of dose planning and delivery for both uniform and positron emission tomography-computed tomography (PET-CT) based heterogeneous dose distributions. We employed a syngeneic rat rhabdomyosarcoma model, which was irradiated by volumetric modulated arc therapy (VMAT) with uniform or heterogeneous 6 MV photon dose distributions. Mean dose to the gross tumor volume (GTV) as a whole was kept at 12 Gy for all treatment arms. For the nonuniform plans, the dose was redistributed to treat the 30% of the GTV representing the biological target volume (BTV) with a dose 40% higher than the rest of the GTV (GTV – BTV) (~15 Gy was delivered to the BTV vs. ~10.7 Gy was delivered to the GTV – BTV). Cone beam computed tomography (CBCT) images acquired for each rat prior to irradiation were used to correctly reposition the tumor and calculate the delivered 3D dose. Film quality assurance was performed using a water-equivalent rat phantom. A comparison between CT or CBCT doses and film measurements resulted in passing rates >98% with a gamma criterion of 3%/2 mm using 2D dose images. Moreover, between the CT and CBCT calculated doses for both uniform and heterogeneous plans, we observed maximum differences of <2% for mean dose to the tumor and mean dose to the biological target volumes. In conclusion, we have developed a robust method for dose painting in a rat tumor model on a clinical platform, with a high accuracy achieved in the delivery of complex dose distributions. Our work demonstrates the technical feasibility of this approach and enables future investigations on the therapeutic effect of preclinical dose painting strategies using a state-of-the-art clinical platform.

High dose rate and flattening filter free irradiation can be safely implemented in clinical practice.

Purpose: We hypothesize that flattening filter free (FFF) high dose rate irradiation will decrease cell survival in normal and cancer cells with more pronounced effects in DNA repair deficient cells. Additionally, we hypothesize that removal of the flattening filter will result in an enhanced relative biological effectiveness independent of the dose rate. Materials and methods: Clonogenic survival was assessed after exposure to dose rates of 4 or 24 Gy/min (FFF 10 megavolt [MV] photon beam) using a Varian TrueBeam accelerator. Additionally, cells were exposed to 4 Gy/min with or without flattening filter. Relative biological effectiveness estimations were performed comparing the different beam photon spectra. Results: Cell survival in tumor and normal cell lines was not influenced by high dose rate irradiation. The intrinsic radiation sensitivity of DNA repair deficient cells was not affected by high dose rate compared to normal dose rate. Furthermore, the relative biological effectiveness was not significantly different from unity in any of the cell lines for both FFF and conventional flattened beam exposures. Conclusions: High dose rate irradiation did not affect long-term survival and DNA repair for cell lines of different tissues. This suggests that high dose rate does not influence treatment outcome or treatment toxicity and could be safely implemented in clinical routine.

PO-0785 : Inverse planning of beam-on times for precision image-guided 3D small animal radiotherapy treatments

PO-0785 Inverse planning of beam-on times for precision imageguided 3D small animal radiotherapy treatments M. Balvert, S.J. Van Hoof, P.V. Granton, D. Trani, D. Den Hertog, A.L. Hoffmann, F. Verhaegen Center for Economic Research (CentER) Tilburg University, Econometric and Operations Research, Tilburg, The Netherlands Maastricht Radiation Oncology (MAASTRO Clinic), Physics Research, Maastricht, The Netherlands Purpose/Objective: Advances in small animal radiotherapy enable the delivery of increasingly complex heterogeneous dose distributions on the millimeter scale, but methods to plan complicated small animal treatments remain in their infancy. A pre-clinical irradiation plan is usually created based on cone beam CT data with the animal in treatment position under anesthesia. Combined with demands on throughput, fast and easy treatment planning methods and algorithms are required. The purpose of this study is to develop an optimization model that determines beam-on times for a given beam configuration, and to assess the benefits of automated treatment planning for small animal radiotherapy. Materials and Methods: The applied model determines a Pareto-optimal solution based on user-provided weights for objectives. An interactive approach allows the user to select the plan that yields the most preferred trade-offs. Two cases based on cone beam CT data of a rat were used, and manual and model-based optimization results were compared using dose-volume metrics. The kidneys, spine and gastrointestinal tract (GI) were delineated as organs at risk (OARs) and a fictitious planning target volume (PTV) was created around the spine. In case 1, the left kidney was targeted as PTV with four 10x10 mm beams and for case 2, twelve 8x10 mm beams were used to target the PTV around the spine. A PTV dose of 8 Gy was prescribed, with a mean dose between 8 and 10 Gy as constraint. Differences between prescribed and planned PTV dose, as well as OAR doses were included in penalty objectives. The model was integrated in a research version of Monte Carlo based small animal treatment planning system SmART-Plan (v2.0 Precision X-ray). Results: Results show that manual and automated treatment planning yields plans of similar quality as shown in the figure and table. A similar amount of time was needed for manual and model-based optimization. In this period, manual optimization generates a single plan, while a set of Paretooptimal plans is created with automated optimization, allowing for a more substantiated choice on trade-offs. Automated optimization often uses fewer beams than manual optimized plans, therewith lowering treatment delivery time. Additional benefits of automated planning include a decreased dependence on the planning skills of the user (often absent in pre-clinical research), and the potential to improve treatment standardization among institutions. For more complex irradiations, manual planning becomes infeasible, making automation a necessity.

Abstract 4422: Activation of beta-catenin/TCF-4 signaling leading to intestinal tumorigenesis in APCMin/+ mice is radiation quality dependent

Purpose: Radiation quality depends on loss of energy in tissues per unit distance travelled and could be low-linear energy transfer (low-LET) or high-linear energy transfer (high-LET). The low-LET radiation like gamma-rays and x-rays loose less and hence deposits less energy in tissues and are sparsely ionizing. However, the high-LET radiation like α-particle, neutron, and heavy ions loose more and so deposits more energy in tissues traversed. Epidemiological studies showed strong correlation between radiation exposure and colorectal cancer (CRC) and CRC is the second leading cause of cancer mortality in the USA. However, it is not known if activation of molecular pathways involved in CRC and consequent intestinal tumorigenesis is radiation quality dependent. Methods: Female APCMin/+ mice (6-8 weeks) were irradiated with 5 Gy of gamma radiation, a tumorigenic dose in these mice. After 90 days mice were euthanized, intestinal tumors counted and tumor samples were flash frozen. Tumor samples were subjected to histology, western blot and immunohistochemistry (IHC) to dissect the APC/β-catenin/TCF4 pathway, commonly altered in human CRC. Results of gamma radiation were compared to two qualitatively different types of radiation - high-energy proton (energy: 1 giga electron volt (GeV)) and iron (56-Fe) ion (energy: 1 GeV/nucleon). We used 4 Gy of 56-Fe and 4.7 Gy of proton, which are isotoxic to the gamma radiation dose of 5 Gy determined using a quality factor 1.25 and 1.06 respectively. Quantitation of IHC and western blot were performed using ImageJ v2.45 softwere. Results: Quantitative observations showed enhanced intestinal tumorigenesis after exposure to proton and 56-Fe radiation. Marked increases in β-catenin and TCF4 were associated with a decreased phospho-β-catenin and increased phospho-GSK3β in proton and 56-Fe mice. Increased β-catenin was also associated with higher levels of cyclin-D1 and c-myc in these samples. Furthermore, compared to gamma radiation, strikingly lower level of p53 along with higher MDM2 was observed in proton and 56-Fe samples. Interestingly, however, the decrease in p53 and increase in MDM2 was higher in 56-Fe. These observations were further supported by IHC staining of tumor sections. Statistical analyses to support these observations in proton and 56-Fe compared to ≤ ray or spontaneous tumors will be presented. Conclusions: Considering higher active β-catenin and inactivated GSK3β, we speculate that exposure to high-LET radiation caused stabilization of oncogenic β-catenin and upregulation of c-myc and cyclin-D1 leading to higher tumorigencity. Enhanced potential of high-LET radiation for carcinogenesis is further supported by downregulation of p53. In conclusion, this is the first evidence showing greater potential of high-LET radiation for intestinal carcinogenesis mediated via activation of β-catenin/TCF4 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4422. doi:1538-7445.AM2012-4422