Intracavitary Electron FLASH Radiotherapy in a Canine Cancer Patient With Oral Malignant Melanoma

PURPOSE/OBJECTIVE(S): Studies of electron FLASH radiotherapy (FLASH-RT) in companion animals are being conducted at several institutions. High energy electron beams are generally suitable for treatment of superficial cancers, but of limited use for deep-seated tumors. In this case report, the feasibility of intracavitary electron FLASH-RT is demonstrated.MATERIALS/METHODS: A canine cancer patient with a large oral malignant melanoma in the caudal part of the hard palate was palliatively treated with FLASH-RT at a modified clinical linear accelerator, using a cylindrical PMMA applicator (length 200 mm, inside diameter 50 mm). The patient had a poor prognosis, with an estimated survival of two weeks and no other available treatment options. A dose of 35 Gy was delivered to the depth of dose maximum in 26 pulses with a pulse dose rate of 4.5•105 Gy/s. The average dose rate was 280 Gy/s, corresponding to a total beam-on time of 125 months. One month after the treatment, the tumor was re-irradiated, using the same treatment parameters. The treatment head of the LINAC was fitted with a short electron applicator holder with a Cerrobend collimator at 65 cm from the source. The cylindrical PMMA applicator was aligned perpendicularly to the Cerrobend collimator using soft docking with a 1 cm gap. Prior to the treatments, the 2D dose distribution of the FLASH beam exiting the PMMA applicator was measured with radiographic film. In addition, the dosimetric effect of misalignment between the collimator and the electron applicator was studied based on intentional misalignments of 2.5°, 5°, 2.5 mm, and 5 mm.RESULTS: A partial response of the tumor and clinical improvement of the patient was observed two weeks after the first treatment. A hypopigmented area in the hard palate was observed, consistent with a grade 1 adverse event, but there were no signs of mucositis or reports of patient discomfort. However, due to the thickness of the tumor (> 3 cm), the posterior part only received a limited dose, and the tumor was still obstructing the airflow at one month post treatment. At this time, a second treatment was administered with limited normal tissue receiving full dose from both treatments. At one month post the second treatment, a partial response was achieved, and no evidence of side effects other than hypopigmentation was observed. The patient was euthanized 2.5 months after the initial therapy due to progressive disease involving the oropharynx. The dosimetric evaluation showed that a conformal dose distribution could be obtained by delivering the electron beam using a cylindrical PMMA applicator. Misalignments between the applicator and the collimator were shown to result in reduced dose homogeneity.CONCLUSION: This case report of a canine cancer patient, with a large oral malignant melanoma in the caudal part of the hard palate, has demonstrated the feasibility of intracavitary electron FLASH-RT. Two treatments of 35 Gy each, resulted in prolonged survival and increased quality-of-life

Establishment and Initial Experience of Clinical FLASH Radiotherapy in Canine Cancer Patients

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

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer

Rhizopine biosensors for plant-dependent control of bacterial gene expression.

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal