Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Update in Veterinary Radiation Oncology: Focus on Stereotactic Radiation Therapy.

Stereotactic radiotherapy (SRT) involves the precise delivery of highly conformal, dose-intense radiation to well-demarcated tumors. Special equipment and expertise are needed, and a unique biological mechanism distinguishes SRT from other forms of external beam radiotherapy. Families find the convenient schedules and minimal acute toxicity of SRT appealing. Common indications in veterinary oncology include nasal, brain, and bone tumors. Many other solid tumors can also be treated, including spinal, oral, lung, heart-base, liver, adrenal, and prostatic malignancies. Accessibility of SRT is improving, and new data are constantly emerging to define parameters for appropriate case selection, radiation dose prescription, and long-term follow-up.

Dysregulated miRNAs in a canine model of haemangiosarcoma metastatic to the brain.

Haemangiosarcoma is a highly metastatic and lethal cancer of blood vessel-forming cells that commonly spreads to the brain in both humans and dogs. Dysregulations in phosphatase and tensin (PTEN) homologue have been identified in various types of cancers, including haemangiosarcoma. MicroRNAs (miRNAs) are short noncoding single-stranded RNA molecules that play a crucial role in regulating the gene expression. Some miRNAs can function as oncogenes or tumour suppressors, influencing important processes in cancer, such as angiogenesis. This study aimed to investigate whether miRNAs targeting PTEN were disrupted in canine haemangiosarcoma and its corresponding brain metastases (BM). The expression levels of miRNA-10b, miRNA-19b, miRNA-21, miRNA-141 and miRNA-494 were assessed in samples of primary canine cardiac haemangiosarcomas and their matched BM. Furthermore, the miRNA profile of the tumours was compared to samples of adjacent non-cancerous tissue and healthy control tissues. In primary cardiac haemangiosarcoma, miRNA-10b showed a significant increase in expression, while miRNA-494 and miRNA-141 exhibited downregulation. Moreover, the overexpression of miRNA-10b was retained in metastatic brain lesions. Healthy tissues demonstrated significantly different expression patterns compared to cancerous tissues. In particular, the expression of miRNA-10b was nearly undetectable in both control brain tissue and perimetastatic cerebral tissue. These findings can provide a rationale for the development of miRNA-based therapeutic strategies, aimed at selectively treating haemangiosarcoma.

Glioma with cribriform plate involvement in 6 dogs.

Distinct patterns of local infiltration are a common feature of canine oligodendroglioma and astrocytoma, and typically involve the surrounding neuroparenchyma, ventricles, or leptomeninges. Infiltration of adjacent extraneural sites is rare and has not been well documented in veterinary medicine. Here we describe 6 canine gliomas with cribriform plate involvement (compression or infiltration) and caudal nasal invasion confirmed by neuroimaging, autopsy, and/or histology. All affected dogs were adults (9-12-y-old), and 3 were brachycephalic. Clinical signs were associated with the brain tumor, with no respiratory signs reported. Magnetic resonance imaging in 2 patients revealed a rostral intraparenchymal telencephalic mass with extension into the cribriform plate. All dogs were euthanized. Gross changes consisted of poorly demarcated, white or pale-yellow, soft, and, in oligodendrogliomas, gelatinous, intraparenchymal masses that expanded the rostral portions of the telencephalon and adhered firmly to the ethmoid bone and cribriform plate. Gliomas were classified as high-grade oligodendrogliomas (4 cases) and high-grade astrocytomas (2 cases) based on histology and immunohistochemistry for OLIG2 and GFAP. In all cases, there was evidence of cribriform plate invasion and, in one case, additional invasion of the caudal nasal cavity.

Anaplastic oligodendroglioma with nasal invasion and systemic metastasis in a dog.

An 11-year-old spayed female French bulldog was referred on suspicion of nasal tumor. Anaplastic oligodendroglioma in the olfactory bulb that was suspected to have invaded the nasal cavity was diagnosed from imaging and histopathology. Metastasis to cervical lymph nodes was suspected, with no other metastases identified. The brain-to-nasal lesion and lymph nodes were treated with hypo-fractionated radiation therapy. Nasal congestion soon resolved. About 3 months later, follow-up computed tomography revealed multiple hepatic and splenic masses, which were cytologically suspected as metastatic oligodendroglioma. Nimustine, followed by toceranib phosphate, seemed to have no effect, and the dog died on day 167. Postmortem examination revealed the primary tumor disappearance and systemic metastases. Canine oligodendroglioma can grow outside the cranial vault, and systemically metastasize.

Meningioma and glioma as the first collision brain tumour reported in small animals.

We describe the clinical and pathological features of a brain collision tumour consisting of a fibrous meningioma and an anaplastic oligoastrocytoma in a 14-year-old male neutered French Bulldog. Computed tomography of the brain revealed a poorly defined, intra-axial lesion affecting the left frontal lobe. Following euthanasia, histological examination showed an anaplastic oligoastrocytoma invading the brain parenchyma and an adjacent fibrous meningioma. As synchronous intracranial tumours are rare in animals, the aims of this report are to describe the clinical, imaging and histopathological features of an intracranial collision tumour in a dog and highlight the importance of a complete histopathological study despite the imaging findings.

Efficacy of stereotactic radiation therapy for the treatment of confirmed or presumed canine glioma.

Intracranial gliomas are the second most common brain tumour in dogs. Radiation therapy provides a minimally invasive treatment option for this tumour type. Earlier publications reporting on the use of non-modulated radiation therapy suggested a poor prognosis for dogs with glioma, with median survival times ranging between 4 and 6 months; more recent literature utilizing stereotactic radiation therapy (SRT) demonstrates that the prognosis for canine gliomas may be more promising, with survival times closer to 12 months. A single institution retrospective study was performed between 2010 and 2020 investigating the outcomes of dogs with biopsy-confirmed glioma or a presumptive diagnosis of intra-cranial glioma based on MRI characteristics that were treated with SRT. Twenty-three client-owned dogs were included. Brachycephalic breeds were overrepresented, totalling 13 dogs (57%). SRT protocols included 16 Gy single fraction (n = 1, 4%), 18 Gy single fraction (n = 1, 4%), 24 Gy in 3 daily fractions (n = 20, 91%), or 27 Gy in four daily fractions (n = 1, 4%). Twenty-one dogs (91%) had improvement of their presenting clinical signs following SRT. Median overall survival time (MST) was 349 days (95% CI, 162-584). Median disease specific survival time was 413 days (95% CI, 217-717). When SRT is incorporated into the management plan for dogs with confirmed or presumed intracranial glioma, a median survival time of approximately 12 months may be achievable.

A case of nerve sheath tumor followed by multicentric high-grade T-cell lymphoma in an African pygmy hedgehog (Atelerix albiventris).

A 3-year-old intact male African pygmy hedgehog was presented at the Teaching Hospital of the Faculty of Veterinary Medicine, University of Belgrade, with a growth on the left side of its abdomen. After clinical examination, the mass was surgically removed, and histopathological findings indicated a nerve sheath tumor. The hedgehog fully recovered after surgery and was euthanized eight months later due to the appearance of multicentric changes in the internal organs. Further necropsy and macroscopic, cytologic, histopathologic, and immunohistochemical findings revealed that the tumor was a multicentric high-grade T-cell lymphoma. This is an unusual case of an African pygmy hedgehog with two different neoplasms-a nerve sheath tumor followed by lymphoma.

Establishment and characterization of two novel patient-derived lines from canine high-grade glioma.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

The T2-FLAIR mismatch sign as an imaging biomarker for oligodendrogliomas in dogs.

BACKGROUND: In humans, the T2-weighted (T2W)-fluid-attenuated inversion recovery (FLAIR) mismatch sign (T2FMM) is a specific imaging biomarker for the isocitrate dehydrogenase 1 (IDH1)-mutated, 1p/19q non-codeleted low-grade astrocytomas (LGA). The T2FMM is characterized by a homogeneous hyperintense T2W signal and a hypointense signal with a hyperintense peripheral rim on FLAIR sequences. In gliomas in dogs, the T2FMM has not been described. HYPOTHESES/OBJECTIVES: In dogs with focal intra-axial brain lesions, T2FMM will discriminate gliomas from other lesions. The T2FMM will be associated with the LGA phenotype and presence of microcysts on histopathology. Interobserver agreement for T2FMM magnetic resonance imaging (MRI) features will be high. ANIMALS: One hundred eighty-six dogs with histopathologically diagnosed focal intra-axial lesions on brain MRI including oligodendrogliomas (n = 90), astrocytomas (n = 47), undefined gliomas (n = 9), cerebrovascular accidents (n = 33), and inflammatory lesions (n = 7). METHODS: Two blinded raters evaluated the 186 MRI studies and identified cases with the T2FMM. Histopathologic and immunohistochemical slides of T2FMM cases were evaluated for morphologic features and IDH1-mutations and compared to cases without the T2FMM. Gene expression analyses were performed on a subset of oligodendrogliomas (n = 10) with and without T2FMM. RESULTS: The T2FMM was identified in 14/186 (8%) of MRI studies, and all dogs with T2FMM had oligodendrogliomas (n = 12 low-grade [LGO], n = 2 high-grade [HGO]; P < .001). Microcystic change was significantly associated with the T2FMM (P < .00001). In oligodendrogliomas with T2FMM, IDH1-mutations or specific differentially expressed genes were not identified. CONCLUSION AND CLINICAL IMPORTANCE: The T2FMM can be readily identified on routinely obtained MRI sequences. It is a specific biomarker for oligodendroglioma in dogs, and was significantly associated with non-enhancing LGO.

Machine learning predicts histologic type and grade of canine gliomas based on MRI texture analysis.

Conventional MRI features of canine gliomas subtypes and grades significantly overlap. Texture analysis (TA) quantifies image texture based on spatial arrangement of pixel intensities. Machine learning (ML) models based on MRI-TA demonstrate high accuracy in predicting brain tumor types and grades in human medicine. The aim of this retrospective, diagnostic accuracy study was to investigate the accuracy of ML-based MRI-TA in predicting canine gliomas histologic types and grades. Dogs with histopathological diagnosis of intracranial glioma and available brain MRI were included. Tumors were manually segmented across their entire volume in enhancing part, non-enhancing part, and peri-tumoral vasogenic edema in T2-weighted (T2w), T1-weighted (T1w), FLAIR, and T1w postcontrast sequences. Texture features were extracted and fed into three ML classifiers. Classifiers' performance was assessed using a leave-one-out cross-validation approach. Multiclass and binary models were built to predict histologic types (oligodendroglioma vs. astrocytoma vs. oligoastrocytoma) and grades (high vs. low), respectively. Thirty-eight dogs with a total of 40 masses were included. Machine learning classifiers had an average accuracy of 77% for discriminating tumor types and of 75.6% for predicting high-grade gliomas. The support vector machine classifier had an accuracy of up to 94% for predicting tumor types and up to 87% for predicting high-grade gliomas. The most discriminative texture features of tumor types and grades appeared related to the peri-tumoral edema in T1w images and to the non-enhancing part of the tumor in T2w images, respectively. In conclusion, ML-based MRI-TA has the potential to discriminate intracranial canine gliomas types and grades.

Quantitative analysis of magnetic resonance images for characterization of blood-brain barrier dysfunction in dogs with brain tumors.

BACKGROUND: Blood-brain barrier (BBB) permeability can be assessed quantitatively using advanced imaging analysis. HYPOTHESIS/OBJECTIVES: Quantification and characterization of blood-brain barrier dysfunction (BBBD) patterns in dogs with brain tumors can provide useful information about tumor biology and assist in distinguishing between gliomas and meningiomas. ANIMALS: Seventy-eight hospitalized dogs with brain tumors and 12 control dogs without brain tumors. METHODS: In a 2-arm study, images from a prospective dynamic contrast-enhanced (DCE; n = 15) and a retrospective archived magnetic resonance imaging study (n = 63) were analyzed by DCE and subtraction enhancement analysis (SEA) to quantify BBB permeability in affected dogs relative to control dogs (n = 6 in each arm). For the SEA method, 2 ranges of postcontrast intensity differences, that is, high (HR) and low (LR), were evaluated as possible representations of 2 classes of BBB leakage. BBB score was calculated for each dog and was associated with clinical characteristics and tumor location and class. Permeability maps were generated, using the slope values (DCE) or intensity difference (SEA) of each voxel, and analyzed. RESULTS: Distinctive patterns and distributions of BBBD were identified for intra- and extra-axial tumors. At a cutoff of 0.1, LR/HR BBB score ratio yielded a sensitivity of 80% and specificity of 100% in differentiating gliomas from meningiomas. CONCLUSIONS AND CLINICAL IMPORTANCE: Blood-brain barrier dysfunction quantification using advanced imaging analyses has the potential to be used for assessment of brain tumor characteristics and behavior and, particularly, to help differentiating gliomas from meningiomas.

A review of primary central nervous system neoplasms of cats.

Primary central nervous system (CNS) neoplasms are uncommonly diagnosed in cats. The majority of primary feline CNS neoplasms described in the veterinary literature consist of meningioma and glioma occurring mainly in the brain and less often in the spinal cord. Although most neoplasms can be diagnosed based on routine histologic evaluation, less typical tumors need to be further characterized using immunohistochemistry. This review compiles the relevant information about the most common primary CNS neoplasms of cats available in the veterinary literature, aiming to serve as a converging source of information for the topic.

Susceptibility artifact morphology is more conspicuous on susceptibility-weighted imaging compared to T2* gradient echo sequences in the brains of dogs and cats with suspected intracranial disease.

Susceptibility-weighted imaging (SWI) has been found to be more reliable in the detection of vessels and blood products than T2*-weighted gradient echo (GE) in several human brain diseases. In veterinary medicine, published information on the diagnostic usefulness of SWI is lacking. The aim of this retrospective observational study was to investigate the value of SWI compared to T2*-weighted GE images in a population of dogs and cats with presumed, MRI-based diagnoses grouped as neoplastic (27), cerebrovascular (14), inflammatory (14), head trauma (5), other pathologies (4), or that were normal (36). Areas of signal void (ASV) were assessed based on shape, distribution, number, and conspicuity. Presence of ASV was found in 31 T2*-weighted GE and 40 SWI sequences; the conspicuity of lesions increased in 92.5% of cases with SWI. A 44.7% increase in the number of cerebral microbleeds (CMBs) was identified within the population using SWI (110) compared to T2*-weighted GE (76). Linear ASV presumed to be abnormal vascular structures, as are reported in humans, were identified in 12 T2*-weighted GE and 19 SWI sequences. In presumed brain tumors, abnormal vascular structures were detected in 11 of 27 (40.7%) cases on T2*-weighted GE and in 16 of 27 (59.3%) cases on SWI, likely representing tumor neovascularization; amorphous ASV interpreted as presumed hemorrhages on T2*-weighted GE were diagnosed as vessels on SWI in five of 27 (18.5%) cases. Since SWI shows ASV more conspicuously than T2*-weighted GE, the authors advocate the use of SWI in veterinary patients.

Doublecortin immunolabeling in canine gliomas with distinct degrees of tumor infiltration.

Increased doublecortin (DCX) immunolabeling at the tumor margins has been associated with tumor infiltration in human glioma and canine anaplastic meningioma. No association between DCX immunolabeling and glioma infiltration has been reported in dogs, to our knowledge. Here we compare the DCX immunolabeling in 14 diffusely infiltrating gliomas (gliomatosis cerebri) and 14 nodular gliomas with distinct degrees of tumor infiltration. Cytoplasmic DCX immunolabeling was classified according to intensity (weak, moderate, strong), distribution (1 = <30% immunolabeling, 2 = 30-70% immunolabeling, 3 = >70% immunolabeling), and location within the neoplasm (random or at tumor margins). Immunolabeling was detected in 6 of 14 (43%) diffusely infiltrating gliomas and 8 of 14 (57%) nodular gliomas. Diffusely infiltrating gliomas had moderate and random immunolabeling, with distribution scores of 1 (4 cases) or 2 (2 cases). Nodular gliomas had strong (6 cases) or moderate (2 cases) immunolabeling, with distribution scores of 1 (3 cases), 2 (3 cases), and 3 (2 cases), and random (6 cases) and/or marginal (3 cases) immunolabeling. Increased DCX immunolabeling within neoplastic cells palisading around necrosis occurred in 4 nodular gliomas. DCX immunolabeling was not increased at the margins of diffusely infiltrating gliomas, indicating that DCX should not be used as an immunomarker for glioma infiltration in dogs.

Immunohistochemical study of neural stem cell lineage markers in canine brains, gliomas, and a glioma cell line.

Neural stem cells (NSCs) produce neuron intermediate progenitor cells (nIPC), oligodendrocyte precursor cells (OPCs), and immature astrocytes. To confirm NSC lineages in the normal canine brain and the association of these cells with gliomas, an immunohistochemical study was conducted on fetal and adult canine brains, gliomas, and a glioma cell line. In fetal brains, glial fibrillary acidic protein (GFAP)- and nestin-immunolabeled NSC were observed in the ventricular zone, ß-3 tubulin- and/or neuronal nuclei (NeuN)-immunolabeled nIPC in the subventricular zone (SVZ), and platelet-derived growth factor receptor-α (PDGFR-α)- and OLIG2-immunolabeled OPC and GFAP- and OLIG2-immunolabeled immature astrocytes in the SVZ and intermediate zone. Ki-67 immunohistochemistry revealed that nIPC exhibited high proliferative activity. Quiescent nIPC and OPC were observed in adult brains. Among 58 glioma cases including 4 low-grade oligodendrogliomas (LGOGs), 48 high-grade oligodendrogliomas (HGOGs), 1 low-grade astrocytoma, and 5 high-grade astrocytomas (HGACs), immunohistochemical analyses revealed that oligodendrogliomas expressed PDGFR-α and OLIG2, whereas astrocytomas expressed GFAP and OLIG2. HGOG showed significantly higher immunohistochemical scores for NeuN and ß-3 tubulin than LGOG. The Ki-67 labeling index was high in PDGFR-α and NeuN-immunolabeled tumor cells, and low in ß-3 tubulin- and synaptophysin-immunolabeled cells. A HGOG cell line possessed the same immunohistochemical characteristics as HGOG. In this study, glioma cells with the OPC and IPC immunophenotypes had a higher Ki-67 labeling index, indicating their high proliferative activity. Furthermore, high-grade gliomas showed the characteristics of nIPC and neurons, which may suggest the pluripotent NSC lineage nature of these tumors.

CSF from a puppy with a cerebral vascular hamartoma.

A 9-week-old puppy with refractory seizures and a dome-shaped head presented to the Mississippi State College of Veterinary Medicine Specialty Center for suspected hydrocephalus. Computerized tomography (CT) findings included transtentorial herniation and an intra-axial mass with dystrophic mineralization. Cerebrospinal fluid analysis revealed an increased nucleated cell count of 1100/µl (RI < 5/µl), erythrocyte count of 2.2 × 106 /µl, and markedly increased microprotein of 1939 mg/dl (RI < 30 mg/dl). On cytologic examination of the CSF, numerous erythrophagocytic, and hemosiderin-laden macrophages were observed, which indicated chronic active hemorrhage. Many neutrophils, macrophages, and lymphocytes that contained numerous intracytoplasmic, pleomorphic, bright yellow crystals were observed. Considering the ongoing hemorrhage, the crystals were presumed to be hematoidin. A biopsy with histopathology was performed on the intra-axial mass, and the results were consistent with a vascular hamartoma. We speculate that the formation of these crystals was related to the ongoing hemorrhage associated with the vascular hamartoma. Identification of these crystals may be useful to aid in the identification of chronic hemorrhage associated with vascular malformations or lesions within the central nervous system.

Grading of oligodendroglioma in dogs based on magnetic resonance imaging.

BACKGROUND: Oligodendroglioma (OG) accounts for 22% of primary brain tumors in dogs. Oligodendroglioma in dogs is graded as low-grade (II) or high-grade (III), based on the presence of microvascular proliferation and necrosis. OBJECTIVE: To investigate if magnetic resonance imaging (MRI) features differ between OG II and III in dogs. ANIMALS: Thirty-two dogs with histological diagnosis of intracranial OG and MRI. METHODS: Retrospective descriptive study. Histology was reviewed to grade OG according to the revised classification. Brain MRI results were reviewed following criteria including contrast enhancement (CE) pattern, presence of cystic structures, gradient-recalled-echo (GRE) signal voids, and necrosis based on signal intensity, as well as diffusion-weighted imaging characteristics. The MRI features were compared between OG II and III using Fisher's exact tests and logistic regression models. RESULTS: Histology identified 8 dogs with OG II (25%) and 24 with OG III (75%). All OG III showed moderate-to-marked CE including 18/24 (75%) with a ring pattern. These features were not seen in OG II. Heterogeneity, cystic structures, GRE signal voids, and necrosis were associated with OG III. No difference in diffusion characteristics was detected between OG II and III. CONCLUSION AND CLINICAL IMPORTANCE: Moderate-to-marked CE and ring pattern were present in dogs with OG III but not in OG II. The presence of cystic structures, GRE signal voids, and necrosis was strongly associated with OG III. Although the importance of brain tumor grading in dogs with regard to prognosis and treatment options remains unknown, the results indicate that MRI reflects the histological features used for grading OG in dogs.

Treatment of intracranial neoplasia in dogs using higher doses: A randomized controlled trial comparing a boosted to a conventional radiation protocol.

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.