FLASH radiotherapy has emerged as a treatment technique with great potential to increase the differential effect between normal tissue toxicity and tumor response compared to conventional radiotherapy. To evaluate the feasibility of FLASH radiotherapy in a relevant clinical setting, we have commenced a feasibility and safety study of FLASH radiotherapy in canine cancer patients with spontaneous superficial solid tumors or microscopic residual disease, using the electron beam of our modified clinical linear accelerator. The setup for FLASH radiotherapy was established using a short electron applicator with a nominal source-to-surface distance of 70 cm and custom-made Cerrobend blocks for collimation. The beam was characterized by measuring dose profiles and depth dose curves for various field sizes. Ten canine cancer patients were included in this initial study; seven patients with nine solid superficial tumors and three patients with microscopic disease. The administered dose ranged from 15 to 35 Gy. To ensure correct delivery of the prescribed dose, film measurements were performed prior to and during treatment, and a Farmer-type ion-chamber was used for monitoring. Treatments were found to be feasible, with partial response, complete response or stable disease recorded in 11/13 irradiated tumors. Adverse events observed at follow-up ranging from 3-6 months were mild and consisted of local alopecia, leukotricia, dry desquamation, mild erythema or swelling. One patient receiving a 35 Gy dose to the nasal planum, had a grade 3 skin adverse event. Dosimetric procedures, safety and an efficient clincal workflow for FLASH radiotherapy was established. The experience from this initial study will be used as a basis for a veterinary phase I/II clinical trial with more specific patient inclusion selection, and subsequently for human trials.

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes (64Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.

In the western world, approximately 50% of all cancer patients receive radiotherapy alone or in combination with surgery or chemotherapy. Image-guided radiotherapy (IGRT) has in recent years been introduced to enhance precision of the delivery of radiation dose to tumor tissue. Fiducial markers are often inserted inside the tumor to improve IGRT precision and to enable monitoring of the tumor position during radiation therapy. In the present article, a liquid fi ducial tissue marker is presented, which can be injected into tumor tissue using thin and fl exible needles. The liquid fi ducial has high radioopacity, which allows for marker-based image guidance in 2D and 3D X-ray imaging during radiation therapy. This is achieved by surface-engineering gold nanoparticles to be highly compatible with a carbohydrate-based gelation matrix. The new fi ducial marker is investigated in mice where they are highly biocompatible and stable after implantation. To investigate the clinical potential, a study is conducted in a canine cancer patient with spontaneous developed solid tumor in which the marker is successfully injected and used to align and image-guide radiation treatment of the canine patient. It is concluded that the new fi ducial marker has highly interesting properties that warrant investigations in cancer patients.

Background The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 ( 64 Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor’s EPR level are therefore warranted. Materials and Methods To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64 Cu-liposome PET/CT (EPR effect) and 68 Ga-RGD PET/CT (neoangiogenesis), 18 F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205). Results 64 Cu-liposome and 68 Ga-RGD SUV max displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUV mean , 68 Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.

Cytotoxic drugs encapsulated into liposomes were originally designed to increase the anticancer response, while minimizing off-target adverse effects. The first liposomal chemotherapeutic drug was approved for use in humans more than 20 years ago, and the first publication regarding its use in a canine cancer patient was published shortly thereafter. Regardless, no general application for liposomal cytotoxic drugs has been established in veterinary oncology till now.Due to the popularity of canines as experimental models for pharmacokinetic analyses and toxicity studies, multiple publications exist describing various liposomal drugs in healthy dogs. Also, some evidence for its use in veterinary cancer patients exists, especially in canine lymphoma, canine splenic hemangiosarcoma and feline soft tissue sarcoma, however, the results have not been overwhelming. Reasons for this may be related to inherent issues with the enhanced permeability and retention effect, the tumour phenomenon which liposomal drugs exploit. This effect seems very heterogeneously distributed in the tumour. Also, it is potentially not as ubiquitously occurring as once thought, and it may prove important to select patients for liposomal therapy on an individual, non-histology-oriented, basis. Concurrently, new developments with active-release modified liposomes in experimental models and humans will likely be relevant for veterinary patients as well, and holds the potential to improve the therapeutic response. It, however, does not resolve the other challenges that liposomal chemotherapy faces, and more work still needs to be done to determine which veterinary patients may benefit the most from liposomal chemotherapy. K E Y W O R D Schemotherapy, liposomes, nanomedicine, oncology, small animal