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Abstract Background Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood‐brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour‐specific markers. Objective To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. Methods We evaluated the sensitising effect of CCNU alone and in combination with TMZ‐irradiation in canine glioma J3T‐BG cells and long‐term drug‐exposed subclones by using clonogenic survival and proliferation assays. Bisulphite‐SEQ and Western Blot were used to investigate molecular alterations. Results TMZ (200 μM) or CCNU alone (5 μM) reduced the irradiated survival fraction (4 Gy) from 60% to 38% (p = 0.0074) and 26% (p = 0.0002), respectively. The double‐drug combination reduced the irradiated survival fraction (4 Gy) more potently to 12% (p < 0.0001). After long‐term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU‐resistant cells, both, single‐drug CCNU (p = 0.0006) and TMZ (p = 0.0326) treatment combined with irradiation (4 Gy) remained effective. The double‐drug‐irradiation combination reduced the cell survival by 86% (p < 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ‐resistant cells, only the double‐drug combination with irradiation (4 Gy) reduced the cell survival by 88% (p = 0.0057) while single‐drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P‐gp expression while MGMT‐methylation profile analysis showed a general high methylation level in the parental and long‐term treated cell lines. Conclusions Our findings indicate that combining CCNU with TMZ‐irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

Abstract Background Local progression of intracranial tumors can be the consequence of insufficient radiation dose delivered. Dose increases in the brain must be made carefully so as not to risk debilitating adverse effects such as radiation necrosis. Hypothesis A new protocol with 10 × 4 Gy + 11% physical dose increase limited to the macroscopic tumor volume results in a clinically better outcome compared to a 10 × 4 Gy protocol. Animals Fifty‐seven client‐owned dogs with primary intracranial neoplasia. Methods Randomized controlled trial. Twenty‐eight dogs were assigned to the control protocol (10 × 4 Gy) and 29 to the simultaneous integrated boost (SIB) protocol with 4.45 Gy dose increase. Treatment groups were compared for outcome and signs of toxicity. Results Mild, transient acute or early‐delayed adverse radiation effects were observed in 5 dogs. Severe late adverse effects were not seen. Between the protocols, no significant differences were found for outcome (intention‐to‐treat analysis): overall time to progression (TTP) was 708 days (95% confidence interval (95% CI) [545,872]), in the control group it was 828 days (95% CI [401,1256]), and in the SIB group 627 days (95% CI [282,973]; P = .07). Median overall survival (OS) was 684 days (95% CI [516,853]), in the control group it was 724 days (95% CI [623,826]), and in the SIB group 557 days (95% CI [95,1020]; P = .47). None of the tested variables was prognostic in terms of outcome. Conclusion and Clinical Importance The dose escalation used with an 11% physical dose increase did not result in better outcome.

IntroductionA variety of treatment options have been described for canine meningoencephalitis of unknown origin (MUO). Few studies focused on radiation therapy as a second line immunomodulating treatment, implicating its effective use. However, a standard radiation therapy protocol is lacking, and further research will help to evaluate the effect of different dose regimens.MethodsTen dogs diagnosed with MUO based on MRI and CSF findings were prospectively enrolled. The dogs were treated with a shortened whole brain radiation therapy protocol (5 × 4 Gy) in combination with prednisolone. Neurologic changes were quantified using an established scoring scheme. Follow-up MRI and CSF examination was scheduled three months after radiation therapy. Overall survival and time to progression were calculated. Histopathology of the brain was performed in case of death.ResultsSeven dogs were diagnosed de novo and three had a history of relapsing MUO. Neurological status improved in all 10 dogs during radiation therapy, with 4/10 returning to normal shortly after radiation therapy. Three dogs died within the first three months after radiation therapy. At follow-up MRI lesions completely resolved in two dogs, partially resolved in five dogs, and progressed in one dog. After follow-up MRI, dogs were further treated with prednisolone monotherapy (two dogs) and additional immunosuppressant drugs (five dogs). Overall, four dogs showed disease progression, with a mean time to progression of 691 days (95%CI: 396–987) and mean overall survival for all dogs was 723 days (95%CI: 436–1011) (both medians not reached). Histopathology confirmed MUO in three dogs but was suggestive for oligodendroglioma in one dog. Radiation induced side effects were not seen.ConclusionShortened whole-brain radiation therapy could be an additional treatment option for MUO in conjunction to prednisolone, specifically for cases that require rapid relief of symptoms and with relapsing history.

Abstract Background Similar to human glioblastoma patients, glial tumours in dogs have high treatment resistance and a guarded prognosis. In human medicine, the addition of temozolomide to radiotherapy leads to a favourable outcome in vivo as well as a higher antiproliferative effect on tumour cells in vitro. Objectives The aim of the study was to determine the radio‐ and temozolomide‐sensitivity of three canine glial tumour cell lines and to investigate a potential additive cytotoxic effect in combined treatment. Additionally, we wanted to detect the level of MGMT promoter methylation in these cell lines and to investigate a potential association between MGMT promoter methylation and treatment resistance. Methods Cells were treated with various concentrations of temozolomide and/or irradiated with 4 and 8 Gy. Radiosensitization by temozolomide was evaluated using proliferation assay and clonogenic assay, and MGMT DNA methylation was investigated using bisulfite next‐generation sequencing. Results In all tested canine cell lines, clonogenicity was inhibited significantly in combined treatment compared to radiation alone. All canine glial cell lines tested in this study were found to have high methylation levels of MGMT promoter. Conclusions Hence, an additive effect of combined treatment in MGMT negative canine glial tumour cell lines in vitro was detected. This motivates to further investigate the association between treatment resistance and MGMT, such as MGMT promoter methylation status.

Tumor volume is controversially discussed as a prognostic factor in dogs treated with radiation therapy for sinonasal tumors. Dogs' body sizes vary widely and relative rather than absolute tumor volume might provide better prognostic information. Our hypothesis was that relative rather than absolute tumor volume (gross tumor volume, GTV) influences time to progression (TTP) and that a larger tumor volume is correlated with a higher tumor stage. We retrospectively investigated possible correlations of initial GTV to weight, body surface area (BSA), nasal cavity size and the tumor stage in 49 dogs with sinonasal tumors. Here, also presumed sinonasal tumors, esthesioneuroblastomas and histologically benign tumors were included. The possible impact of absolute and relative GTV on response and outcome were assessed according to imaging findings in 34 dogs with available follow-up computed tomographies (CTs) after definitive-intent radiation therapy with either a regular (10x4.2 Gy) or a simultaneously- integrated boost protocol (SIB; GTV boosted to 10x4.83 Gy). In contrast to absolute GTV (p