AAPM WGVRTO report 390: A survey of veterinary radiation oncology equipment and infrastructure in 2022.

Since 2010, there has been little published data on the state of equipment and infrastructure in veterinary radiation oncology clinical practice. These data are important not only to identify the status and use of technology within the veterinary radiation oncology community but also to help identify the extent of medical physics support. The purpose of our study is to report findings from a survey of veterinary radiation oncologists in the USA, Canada, and select centers outside of North America in 2022. A 40-question survey covering topics such as type of radiotherapy equipment, techniques offered, treatment planning systems and dose calculation algorithms, special techniques, board-certified radiation oncologists and residents, and extent of medical physics support was distributed through an online survey tool. Results from 40 veterinary radiation oncology institutions, with equipment explicitly used for veterinary care, suggest that the current state of practice is not dissimilar to what currently exists in human radiation oncology facilities; techniques and technologies commonly employed include flattening filter-free mode megavoltage beams, volumetric arc therapy, daily cone-beam computed tomography, image-guided radiation therapy, and sophisticated dose calculation algorithms. These findings suggest the need for modern radiation oncology acceptance testing, commissioning, and quality assurance programs within the veterinary community. The increase in veterinary radiation oncology residency positions and increasing sophistication of equipment suggests that increased levels of standardized medical physics support would benefit the veterinary radiation oncology community.

Intensity-modulated radiotherapy and chemotherapy for canine right atrial tumors: A retrospective study of seven dogs.

Most primary cardiac tumors in dogs are located in the right atrium/atrial appendage, with hemangiosarcoma being the most common. The aims of this retrospective, case series were to describe outcomes for seven dogs with right atrial tumors treated with hypofractionated intensity-modulated radiotherapy and concurrent vinblastine and propranolol. One dog had a complete response, four dogs had partial responses and two dogs had stable disease after treatment. Effusions resolved in all dogs. Median progression-free survival was 290 days. Five dogs died from metastatic disease, one dog from unrelated neoplasia, and one dog is alive. Median overall survival was 326 days. Three dogs with confirmed hemangiosarcoma survived 244, 326, and 445 days. Two dogs developed clinically significant, but nonfatal, cardiac arrhythmias. One dog that received three courses of radiation had subclinical myocardial and arterial fibrosis at necropsy. Hypofractionated chemoradiotherapy was well tolerated and may provide clinical benefit in dogs with right atrial tumors.

ACVR and ECVDI consensus statement: Reporting elements for toxicity criteria of the veterinary radiation therapy oncology group v2.0.

The toxicity criteria of the veterinary radiation therapy oncology group (VRTOG) version 2 guidelines are a substantial update to reflect significant advances in radiation oncology over the last three decades. Radiation therapy techniques provide precise and spatially accurate radiation delivery, which facilitates treating tumors in more anatomic locations and incorporating hypofractionated protocols. The purpose of this update is to aid radiation oncology teams in capturing and grading clinically relevant data that impacts the decision-making process in everyday practice and the assessment of clinical trials involving radiation therapy. A dedicated committee initially updated the criteria to include more anatomical sites and grades to characterize a broad spectrum of possible radiation-induced acute and late tissue changes. Through the revision process, which solicited and incorporated feedback from all radiation oncologists within the American College of Veterinary Radiology (ACVR) and specialists outside the ACVR, the authors endeavored to create a grading structure reflective of clinical decision-making in daily radiation oncology. The updated VRTOG v2 toxicity criteria guideline complements the updated Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) guidelines. Because radiation oncology continues to progress rapidly, the VRTOG toxicity criteria should be regularly updated as adverse event data that will be collected following this update further informs the practice of radiation oncology.

A prospective, multi-centre, Veterinary Radiation Therapy Oncology Group study reveals potential efficacy of toceranib phosphate (Palladia) as a primary or adjuvant agent in the treatment of canine nasal carcinoma.

Radiation is the standard of care for dogs with nasal tumours. The addition of another therapy that could improve outcome without increasing toxicity is attractive. Medical therapy that could offer better outcome than maximally tolerated dose chemotherapy when radiation therapy (RT) is not possible or is declined is also attractive. This article reports the findings from a prospective, multi-centre, non-randomized, Veterinary Radiation Therapy Oncology Group clinical trial designed to evaluate whether toceranib phosphate (toceranib) has primary activity and if the addition of toceranib to RT could positively impact outcome. Owner's discretion determined enrolment in toceranib alone or toceranib + RT arm. Historical controls for radiation alone were selected from patients treated with identical RT and imaging protocols. Responses were evaluated with pre-treatment and week-16 CT scans. RT total dose of 42 Gy was completed in 10 fractions. Sixty-three dogs enrolled from 10 study sites. Overall response rates (CR + PR) were significantly improved in the toceranib + RT (79.4%) and RT alone (68.9%) arms over toceranib alone (22%) (p = .011). Clinical benefit rates (CR + PR + SD) were significantly improved in the toceranib + RT arm over the RT alone arm at 97.3% and 79.2% respectively (p = .036). Treatment with toceranib alone, toceranib + RT and RT alone resulted in median survival times of 298, 615 and 368 days respectively, but were not statistically significantly different (p = .0502). Adverse events associated with toceranib administration did not potentiate the RT side effect profile. Toceranib appears to have primary activity against nasal carcinoma.

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

RATIONALE: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. METHODS: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. RESULTS: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. CONCLUSIONS: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.

Definitive-intent intensity modulated radiotherapy for modified-Adams' stage 4 canine sinonasal cancer: A retrospective study of 29 cases (2011-2017).

Dogs with sinonasal tumors with cribriform plate lysis (modified Adams' stage 4) treated with non-conformal definitive radiotherapy (RT) have short median survivals of 6-7 months. Intensity-modulated radiotherapy with its greater conformality and tumor dose homogeneity may result in more favorable outcomes. Dogs with epithelial or mesenchymal sinonasal tumors and CT evidence of cribriform lysis that received 10 daily fractions of 4.2 Gray using IMRT by helical tomotherapy were included in this single-institution retrospective case series study. Dogs with distant metastasis, previous treatment, or concurrent chemotherapy were excluded. Based on CT, tumors were divided into two groups: cribriform plate lysis only (stage 4a) or intracranial extension (stage 4b). Twenty-nine dogs were included, 23 with carcinoma and six with sarcoma. Eight dogs had stage 4b tumors; two presented with neurologic signs. Two dogs had lymph node metastasis at diagnosis, one confirmed and one suspected. Radiation dose distributions were standardized and patient positioning for RT was verified daily using on-board megavoltage CT. All evaluable dogs had improvement of clinical signs. Median progression free survival was 177 days (95% CI, 128-294 days). Median overall survival was 319 days (95% CI, 188-499 days). Radiotherapy was well tolerated. The most common side effect was grade 1 or 2 oral mucositis. Two dogs that received additional treatment at progression (stereotactic RT [1]; surgery [1]) developed significant late effects. Image-guided definitive-intent IMRT may improve survival in dogs with modified Adams' stage 4 sinonasal tumors and is associated with low morbidity. Intracranial tumor extension was not prognostic in this cohort of uniformly treated dogs.

Response and outcome following radiation therapy of macroscopic canine plasma cell tumours.

Thirty dogs with macroscopic plasma cell tumours (PCTs) were treated with radiation therapy (RT). Twelve patients were treated with palliative-intent prescriptions (range, 4-10 Gy/fraction (median, 7 Gy/fraction) for a total dose of 20 to 35 Gy (median total dose 30 Gy). Eighteen patients received definitive-intent prescriptions (range, 3.0-4.2 Gy/fraction (median, 3 Gy/fraction) for a total dose of 42 to 54 Gy (median total dose 48 Gy). Involved sites included the oral cavity, skin, multiple myeloma (MM)-associated lytic bone lesions, bone (solitary osseous plasmacytoma; SOP), nasal cavity, larynx, retrobulbar space, lymph node and rectum. Ninety-five percent of evaluable dogs had a complete (CR; 16/22) or partial response (PR; 5/22). Patients with MM experienced significant analgesia. The median progression-free survival (PFS) was 611 days (range: 36-2001 days). Events in the non-MM cases included in-field progression (5/26, 19%) and disseminated disease (5/26, 19%). The median survival time (MST) for all dogs was 697 days (range: 71-2075 days), and when only non-MM cases were considered, MST was 771 days (range: 71-2075 days). Fourteen patients were alive without disease progression or had died of unrelated causes. Achievement of a PR was associated with an inferior PFS and MST as compared with CR. Palliative-intent RT was associated with inferior MST as compared with definitive-intent RT. RT is a useful therapeutic modality for PCTs and tumour responses are often complete and durable, with protracted survivals. The optimal radiation dose and schedule are yet to be defined.

Definitive-intent intensity-modulated radiation therapy provides similar outcomes to those previously published for definitive-intent three-dimensional conformal radiation therapy in dogs with primary brain tumors: A multi-institutional retrospective study.

Radiotherapy with or without surgery is a common choice for brain tumors in dogs. Although numerous studies have evaluated use of three-dimensional conformal radiotherapy, reports of definitive-intent, IMRT for canine intracranial tumors are lacking. Intensity-modulated radiation therapy has the benefit of decreasing dose to nearby organs at risk and may aid in reducing toxicity. However, increasing dose conformity with IMRT calls for accurate target delineation and daily patient positioning, in order to decrease the risk of a geographic miss. To determine survival outcome and toxicity, we performed a multi-institutional retrospective observational study evaluating dogs with brain tumors treated with IMRT. Fifty-two dogs treated with fractionated, definitive-intent IMRT at four academic radiotherapy facilities were included. All dogs presented with neurologic signs and were diagnosed via MRI. Presumed radiological diagnoses included 37 meningiomas, 12 gliomas, and one peripheral nerve sheath tumor. One dog had two presumed meningiomas and one dog had either a glioma or meningioma. All dogs were treated in the macroscopic disease setting and were prescribed a total dose of 45-50 Gy (2.25-2.5 Gy per fraction in 18-20 daily fractions). Median survival time for all patients, including seven cases treated with a second course of therapy was 18.1 months (95% confidence of interval 12.3-26.6 months). As previously described for brain tumors, increasing severity of neurologic signs at diagnosis was associated with a worse outcome. Intensity-modulated radiation therapy was well tolerated with few reported acute, acute delayed, or late side effects.

Multimodality treatment including ONCEPT for canine oral melanoma: A retrospective analysis of 131 dogs.

Canine oral melanoma (OM) is an aggressive cancer with a high rate of metastasis. Surgery and/or radiotherapy (RT) are effective local treatments, yet many dogs succumb to distant metastasis. Immunotherapy represents an attractive strategy for this potentially immunogenic tumor. The objective of this multi-institutional retrospective study was to examine the clinical outcome of dogs with OM treated with ONCEPT melanoma vaccine. Most dogs also underwent surgery and/or RT (8 Gy × four weekly fractions). Dogs with distant metastasis at diagnosis and those receiving concurrent chemotherapy were excluded. One hundred thirty-one dogs treated with ONCEPT were included: 62 had adequate local tumor control defined as complete tumor excision or irradiation of residual microscopic disease; 15 were treated in the microscopic disease setting following an incomplete excision without adjuvant RT; and 54 had gross disease. Median time to progression, median progression-free survival, and median tumor-specific overall survival were 304, 260, and 510 days, respectively. In multivariable analysis, presence of gross disease correlated negatively with all measures of clinical outcome. Other negative prognostic indicators were primary tumor ≥2 cm, higher clinical stage (stages 2 and 3), presence of lymph node metastasis at diagnosis, and caudal location in the oral cavity. Radiotherapy had a protective effect against tumor progression. To date, this is the largest reported series of dogs with OM treated with ONCEPT. Several previously reported prognostic indicators were confirmed.

Definitive-intent radiotherapy for sinonasal carcinoma in cats: A multicenter retrospective assessment.

Treatment of epithelial sinonasal tumours in cats is not commonly reported. In the newer reports, palliative radiation protocols have been described more often than definitive-intent protocols. In this multi-institutional retrospective study, we included 27 cats treated with single-modality radiotherapy. Cats were irradiated using 10 daily fractions of 4.2 Gy. Three cats (11.1%) experienced a complete clinical response and 17 (63%) had a partial clinical response. Stable clinical disease was noted in three cats (11.1%). Four cats (14.8%) showed progression within 3 months following treatment. The median time to progression for all cases was 269 days (95 % confidence intervals [CI]: 225; 314). The proportion of cats free of progression at 1 and 2 years was 24% (95% CI: 22%; 26%) and 5% (95% CI: 5%; 6%), respectively. None of the prognostic factors evaluated were predictive of outcome (anaemia, tumour volume at the time of staging, modified Adams stage, intracranial involvement, facial deformity, epistaxis, inappetence or weight loss). Median overall survival (OS) for all deaths was 452 days (95% CI: 334; 571). The proportion of cats alive at 1 and 2 years was 57% (95% CI: 37%; 77%) and 27% (95% CI: 25%; 29%), respectively. Surprisingly, cats with epistaxis had a longer median OS of 828 days (95% CI: 356; 1301) compared to 296 days (95% CI: 85; 508) in cats without epistaxis, (P = .04, Breslow). Radiation therapy used as a single modality for the treatment of feline sinonasal carcinoma improved clinical signs and was well tolerated but progression within a year was common.

Outcome and failure patterns of localized sinonasal lymphoma in cats treated with first-line single-modality radiation therapy: A retrospective study.

Failure rate and site are not well defined in localized sinonasal lymphoma in cats treated with radiotherapy. In this study, we describe (a) failure pattern, (b) outcome, (c) influence of previously reported prognostic variables on the outcome in cats with suspected localized sinonasal lymphoma. In this multi-institutional retrospective study, we included 51 cats treated with single-modality radiotherapy. Cats were irradiated using 10x4.2Gy (n = 32), 12x3Gy (n = 11) or 5x6Gy (n = 8). Regional lymph nodes were prophylactically irradiated in 24/51 cats (47.1%). Twenty-five cats (49.0%) developed progressive disease: progression was local (nasal) in five (9.8%), locoregional (nodal) in two (3.9%), local and locoregional in three (5.9%), systemic in nine (17.6%) and both local and systemic in six cats (11.8%). No cat receiving prophylactic nodal irradiation had progression in the locoregional lymph nodes. The median time to progression was 974 days (95%CI: 283;1666), with 58% and 53% of cats free of progression at 1 and 2 years, respectively. Median overall survival was 922 days (95%CI: 66;1779) with 61% and 49% alive at 1 and 2 years, respectively. Half of the cats that died of relapse/progression (13/26) died within 6 months of treatment, suggesting possible shortcomings of staging, rapid dissemination of disease or sequential lymphomagenesis. None of the prognostic factors evaluated were predictive of outcome (prednisolone use, anaemia, nasopharyngeal involvement, modified canine Adams tumour stage, protocol, total dose). Radiotherapy is an effective treatment for localized sinonasal lymphoma with a long time to progression. However, in one-third of the cats, systemic disease progression occurs soon after radiotherapy.

Short survival time following palliative-intent hypofractionated radiotherapy for non-resectable canine thyroid carcinoma: A retrospective analysis of 20 dogs.

Radiotherapy is the treatment of choice for non-resectable canine thyroid carcinoma. High tumor response rates and median survival times of 2 years or longer have been previously reported with conventionally fractionated and hypofractionated protocols, even in dogs with distant metastasis. The objective of this retrospective, descriptive, case series study was to evaluate the clinical outcomes of dogs with thyroid carcinoma irradiated with palliative intent using hypofractionated radiotherapy at our institution. Medical records of 20 dogs treated between 1999 and 2014 were reviewed. All dogs had macroscopic primary tumors and presented with tumor-related clinical signs. Median longest tumor diameter was 10 cm. Nineteen dogs (95%) had metastasis (7/19 lymph node; 16/19 distant metastasis). Most dogs were treated with four weekly fractions of 6.5-8 Gy. Radiotherapy was well tolerated in 17 dogs; three died of respiratory compromise before completing radiotherapy. Eleven dogs received adjuvant chemotherapy. Five dogs experienced a local tumor response including two complete and three partial responses. Overall median survival time was 170 days (range, 1-824 days; 95% CI: 58-392 days). Of potential variables examined (radiation delivery system and protocol, tumor size and location, vascular/lymphatic invasion, metastatic disease, chemotherapy, tumor response), only achievement of complete or partial response was predictive of overall survival. In contrast to previously reported cohorts, dogs with clinical signs and stage IV disease predominated in this study. Previous studies may over-estimate survival following hypofractionated radiotherapy in dogs with advanced thyroid carcinoma.

Radiation therapy for intracranial tumours in cats with neurological signs.

OBJECTIVES: The aim of this study was to evaluate the outcome of cats with intracranial tumours presenting with neurological signs treated with radiation therapy. METHODS: This study comprised a retrospective multicentre case series. Medical records of a total of 22 cats with intracranial space-occupying lesions, presenting with neurological signs and/or epileptic seizures and treated with external beam radiation therapy, were reviewed. In the treated cats, patient-, tumour- and treatment-related variables were investigated, including age, sex, tumour location, tumour volume, total radiation dose, equivalent dose in 2 Gy fractions (EQD2), corticosteroid dose, overall treatment time and institution for influence on local tumour control and survival. RESULTS: Based on advanced imaging characteristics, the 22 treated cats presented with meningioma (n = 11), pituitary tumour (n = 8), choroid plexus tumour (n = 2) or glioma (n = 1). Allocated to the neuraxis, 11 lesions were extra-axial, three were intra-axial and eight were located in the pituitary region. At diagnosis, 21 cats exhibited altered neurological status. One cat presented with epileptic seizures and another cat had both seizures and altered neurological status. The mean total physical dose of radiation was 41.63 Gy (± 4.33), range 24-45 Gy. In all but one cat (95.5%), neurological signs improved after radiation therapy. The median progression-free survival was 510 days (95% confidence interval [CI]: 51-969). The proportion free of progression at 1 year was 55.7% (95% CI: 33-78). Fourteen cats died (only in five cases was death related to the intracranial tumour) and eight cats were still alive or lost to follow-up. The median overall survival time was 515 days (95% CI: 66-964). None of the tested variables influenced outcome. CONCLUSIONS AND RELEVANCE: Radiation therapy seems to represent a viable treatment option in cats with intracranial tumours, relieving neurological signs and improving local tumour control. Radiation therapy may be considered for cats with tumours in complicated/inoperable localisations or for cases with a high peri- and postoperative risk.

A survey of stereotactic radiation therapy in veterinary medicine.

Radiotherapy plays an important role in curative and palliative cancer treatment. As a novel radiation delivery technique, stereotactic radiotherapy utilizes three-dimensional-conformal treatment planning, high-precision beam delivery technology, and patient specific position verification to target tumors, often in one to five high-dose fractions. Currently, there is no consensus about best stereotactic radiotherapy practices in veterinary radiotherapy. The objective of this study was to document the breadth of perspectives, techniques, and applications of stereotactic radiotherapy in veterinary medicine. We conducted an online survey of American College of Veterinary Radiology members specializing in radiation oncology to assess how, when, and why stereotactic radiotherapy is being used. Both stereotactic radiotherapy users and nonusers completed the survey. The overall response and survey completion rates were 54% (67/123) and 87% (58/67), respectively. Overall, 55% of respondents reported providing stereotactic radiotherapy at their facility, with a median of 4.5 canine cases and one feline case per month. Delivery methods included C-arm linear accelerator with multi-leaf collimator, helical tomotherapy, and CyberKnife. Nonpituitary intracranial tumors, pituitary tumors, and sinonasal tumors were the most common cancers treated using stereotactic radiotherapy in both species. The most common fractionation scheme was three fractions of 10 Gy/fraction. The results of this survey suggest common availability of stereotactic radiotherapy in veterinary radiation facilities. These results provide valuable information regarding current stereotactic radiotherapy practices in veterinary medicine, and may provide an initial step toward standardizing methods and establishing consensus guidelines.

SINGLE INSTITUTION VARIABILITY IN INTENSITY MODULATED RADIATION TARGET DELINEATION FOR CANINE NASAL NEOPLASIA.

Contouring variability is a significant barrier to the accurate delivery and reporting of radiation therapy. The aim of this descriptive study was to determine the variation in contouring radiation targets and organs at risk by participants within our institution. Further, we also aimed to determine if all individuals contoured the same normal tissues. Two canine nasal tumor datasets were selected and contoured by two ACVR-certified radiation oncologists and two radiation oncology residents from the same institution. Eight structures were consistently contoured including the right and left eye, the right and left lens, brain, the gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV). Spinal cord, hard and soft palate, and bulla were contoured on 50% of datasets. Variation in contouring occurred in both targets and normal tissues at risk and was particularly significant for the GTV, CTV, and PTV. The mean metric score and dice similarity coefficient were below the threshold criteria in 37.5-50% and 12.5-50% of structures, respectively, quantitatively indicating contouring variation. This study refutes our hypothesis that minimal variation in target and normal tissue delineation occurs. The variation in contouring may contribute to different tumor response and toxicity for any given patient. Our results also highlight the difficulty associated with replication of published radiation protocols or treatments, as even with replete contouring description the outcome of treatment is still fundamentally influenced by the individual contouring the patient.

In vitro efficacy of doxorubicin and etoposide against a feline injection site sarcoma cell line.

Feline injection site sarcoma (ISS) is a locally invasive tumor, in which surgical treatment is frequently combined with radiation or chemotherapy to improve tumor control. The focus of this study was to evaluate the cytotoxic effects of doxorubicin or etoposide on a feline injection site sarcoma cell line (JB) and to assess the impact of combining these drugs on cell death and cell cycle. Both single agent and combination drug administration increased cell death and significantly reduced the number of viable cells. Cells in G0/G1 were significantly reduced while the G2/M fraction was significantly increased following treatment. Collectively, combining doxorubicin and etoposide at the lower EC yielded comparable results to the EC50 of either drug alone in degree of cytotoxicity, level of apoptosis, and % of cells in G2/M. The results of this study indicate that doxorubicin and etoposide alone and in combination differentially alter ISS cell viability and cycle.

Influence of different cell storage/culture conditions on spontaneous proliferation and level of tyrosine kinase receptor inhibition in two feline injection-site sarcoma cell lines.

Optimizing cell culture conditions is important when studying cell proliferation and viability, particularly in response to cytotoxic compounds. Altered cell storage conditions can adversely impact proliferation and viability in mortal cell lines. However, little is known regarding the effects on immortal feline cell lines. In the present study, two feline injection-site sarcoma (ISS) cell lines were evaluated under standard culture conditions and three alternative storage/culture conditions for spontaneous proliferation rate and sensitivity to masitinib, a highly selective tyrosine kinase inhibitor with activity against primary and metastatic ISS cell lines. Cell viability was assessed by 7-aminoactinomycin D and cytology. Spontaneous proliferation did not significantly differ across the FBS concentrations (10% vs. 1%) for one cell line, however, with the other cell line spontaneous proliferation was significantly decreased in the 1% FBS 1-step technique, and the cold step technique at both 1% and 10% FBS. When normalized to untreated control cells, the IC50 values for masitinib were comparable across all culture techniques. Furthermore, apoptosis appeared to be the primary mechanism of this proliferation inhibition. Our preliminary findings suggest that select feline sarcoma cell lines cultured in 10% FBS yield comparable cytotoxicity data even when subjected to varying storage/culture conditions.

Roles of four Arabidopsis U-box E3 ubiquitin ligases in negative regulation of abscisic acid-mediated drought stress responses.

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H(2)O(2)) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H(2)O(2) and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H(2)O(2), and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.