DCE-MRI: a review and applications in veterinary oncology.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is a functional imaging technique that assesses the physiology of tumour tissue by exploiting abnormal tumour microvasculature. Advances made through DCE-MRI include improvement in the diagnosis of cancer, optimization of treatment choices, assessment of treatment efficacy and non-invasive identification of prognostic information. DCE-MRI enables quantitative assessment of tissue vessel density, integrity, and permeability, and this information can be applied to study of angiogenesis, hypoxia and the evaluation of various biomarkers. Reproducibility of DCE-MRI results is important in determining the significance of observed changes in the parameters. As improvements are made towards the utility of DCE-MRI and interpreting biologic associations, the technique will be applied more frequently in the study of cancer in animals. Given the importance of tumour perfusion with respect to tumour oxygenation and drug delivery, the use of DCE-MRI is a convenient and powerful way to gain basic information about a tumour.

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours.

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.

Comparison of isodose distributions in canine brain in heterogeneity-corrected versus uncorrected treatment plans using 6 MV photons.

Magnetic resonance (MR) images may be useful for radiation planning due to greater contrast resolution. One disadvantage of MR images for radiation planning is the inability to incorporate electron density information into the dose calculation algorithm. To assess the magnitude of this problem, we evaluated radiation dose distribution in canine brain by comparing computed tomography (CT)-based radiotherapy plans with and without electron density correction. Computerized radiotherapy plans were generated for 13 dogs with brain tumors using 6 MV photons. A tissue-contouring program was used to outline the gross tumor volume (GTV) and the planning target volume (PTV) for each patient. Two treatment plans were generated for each dog. First, the plan was optimized without heterogeneity correction. Then the heterogeneity correction was implemented without changing any other plan parameters. Isodose distributions and dose volume histograms (DVHs) were used to compare the two plans. The D95 (dose delivered to 95% of the volume) within the PTV was calculated for each treatment plan and differences in the D95s were compared. The mean D95s without and with heterogeneity correction were 49.1 +/- 0.7 and 48.9 +/- 1.0Gy, respectively. The absolute mean percent dose difference without and with heterogeneity correction was 1.0 - 0.9% (-1.3-3.2%) and was not considered to be clinically significant. We found no clinically significant difference between CT-based radiotherapy plans without and with heterogeneity correction for brain tumors in small animals, which supports the use of MR-based treatment planning for radiotherapy of small animal brain tumors.

Changes in tumour oxygenation during fractionated hyperthermia and radiation therapy in spontaneous canine sarcomas.

Tumour oxygenation was measured in seven canine soft tissue sarcomas being treated with a fractionated course of radiation and hyperthermia. Measurements obtained during treatment were compared to pre-treatment measurements. The most important finding was an increase in oxygenation in tumours with low pre-treatment oxygenation that persisted throughout treatment. This is an advantageous hyperthermia effect as it may lead to increased radiation cell killing at each fraction. In other tumours, potentially less advantageous changes in oxygenation may be hyperthermia fractionation related and this deserves further investigation.

Epidermal growth factor receptor expression in feline oral squamous cell carcinomas.

Feline oral squamous cell carcinomas (SCC) have a poor prognosis despite aggressive treatment with surgery, radiation and anticancer drugs. Overexpression of the epidermal growth factor receptor (EGFR), a membrane-bound tyrosine kinase receptor, has been found in many human epithelial neoplasms, including oral SCC. EGFR overexpression has been associated with advanced disease and a poor prognosis. The purpose of this study was to determine whether feline oral SCC express EGFR. Thirteen formalin-fixed paraffin wax-embedded biopsy samples from feline oral SCC were analysed for EGFR expression using immunohistochemistry. Nine of 13 tumours (69%) were positive for EGFR expression, suggesting that altered EGFR expression plays a role in feline oral SCC and provides a rationale for a potential clinical benefit using EGFR inhibitors in combination with conventional treatments.

Effect of calcitonin gene related peptide vs sodium nitroprusside to increase temperature in spontaneous canine tumours during local hyperthermia.

The objectives of this study were to compare the effects of two vasodilators, sodium nitroprusside (SNP) and calcitonin gene-related peptide (CGRP) on mean arterial pressure (MAP), heart rate (HR) and temperatures in tumour and surrounding normal tissue during local hyperthermia treatment. Eleven tumour-bearing pet dogs with spontaneous soft tissue sarcomas were given SNP intravenously during local hyperthermia. The drug infusion rate was adjusted to maintain a 20% decrease in MAP. The median (95% CI) increase in the temperature distribution descriptors T(90) and T(50) was 0.2 degrees C (0.0-0.4 degrees C, p = 0.02) and 0.4 degrees C (0.1-0.7 degrees C, p = 0.02), respectively, in tumour. Normal subcutaneous tissue temperatures were mildly increased but remained below the threshold for thermal injury. The effects of CGRP were investigated in six tumour-bearing dogs following a protocol similar to that used for SNP. The median (interquartile (IQ) range) decrease in mean arterial pressure was 19% (15-26%) after CGRP administration and a significant increase was seen in tumour but not normal subcutaneous tissue temperatures. The median (95% CI) increase in the temperature distribution descriptors T(90) and T(50) was 0.5 degrees C (0.1-1.6 degrees C, p = 0.03) and 0.8 degrees C (0.1-1.6 degrees C, p = 0.13), respectively. Administration of SNP or CGRP did not result in local or systemic toxicity in tumour-bearing dogs. However, the magnitude of increase in tumour temperatures was not sufficient to improve the likelihood of increased response rates. Therefore, there is little justification for translation of this approach to human trials using conventional local hyperthermia.

Computed tomographic imaging of infiltrative lipoma in 22 dogs.

Twenty two dogs with an infiltrative lipoma had computed tomographic (CT) images acquired to evaluate the extent of local disease. Ten dogs had undergone at least one cytoreductive surgical procedure (range = 1-3; median = 2) prior to imaging. Twenty dogs had measurable disease on CT images; 2 dogs had diffuse disease at a previous surgical site that could not be measured. Tumor volume (n = 20) ranged from 20 to 5,632 cm3 (median = 345 cm3; mean = 996 cm3). None of the dogs had evidence of bone involvement on the CT images; 2 of the 22 dogs had tumors that did not come into direct contact with osseous structures. All dogs with measurable disease had evidence of a fat opacity mass with variable degrees of muscle infiltration. Eleven of 22 dogs were given intravenous contrast medium prior to image acquisition and there was not evidence of enhancement of the infiltrative lipoma in any dog. Based on CT images, tumors were classified as well-defined in 9 dogs, moderately well-defined in 4, not well-defined in 3 and a mix of well-defined and not well-defined in 6 dogs. Tumors tended to be less well-defined in regions where the infiltrative lipoma interdigitated with normal body fat. It appears CT imaging allows adequate discrimination of tumor with the caveat that differentiation of normal fat from infiltrative lipoma can be problematic.

Retrospective analysis of axial skeleton osteosarcoma in 22 large-breed dogs.

Medical-records of 22 large-breed dogs (>15 kg) with osteosarcoma (OSA) of the axial skeleton were reviewed to determine prevalence of metastasis and survival associated with this neoplasm. All dogs were treated with more than 1 mode of therapy including palliative radiation (n = 12), definitive radiation (n = 8), surgery (n = 7), chemotherapy (n = 12), or some combination of these therapies. Metastasis was documented in 10 of 22 dogs (46%), and the median survival for all dogs was 137 days. Primary cause of death was local tumor recurrence (54%). Breed (retriever versus purebred versus mixed-breed survival was 100, 182, and 264 days, respectively) and radiation therapy protocol (survival in dogs treated with palliative radiation therapy versus those treated with definitive radiation therapy was 79 and 265 days, respectively) were significantly related to survival (P < .05). Prevalence of metastasis and median survival for large-breed dogs with axial skeleton OSA seems to be similar to that reported for large-breed dogs with appendicular skeleton OSA. Definitive radiation therapy may have a role in the treatment of axial skeleton osteosarcoma.

Semiquantitative immunohistochemical analysis for hypoxia in human tumors.

OBJECTIVE: The goal of this study was to develop a semiquantitative scoring system for measuring hypoxia in human tumors by an immunohistochemical marker approach. METHODS AND MATERIALS: Eighteen patients diagnosed with squamous cell carcinoma of the uterine cervix or head and neck were infused intravenously with a solution of pimonidazole hydrochloride at a dose of 0.5 gm/m2. Twenty-four hours later, four biopsies on average from each tumor were fixed in formalin, processed into paraffin blocks, and sectioned. Tissue sections were immunostained for the presence of pimonidazole adducts. Microscopic images (x200) of immunostaining were captured and quantitated by standard image analysis. Images with known amounts of hypoxia spanning ranges of > 0% to 5%, > 5% to 15%, > 15% to 30%, and >30% were assigned scores of +1, +2, +3, and +4, respectively. Three observers then used this calibrated scoring system to analyze hypoxia in tumor sections in a blinded fashion. RESULTS: Excellent interobserver reproducibility was obtained with the calibrated, semiquantitative, immunohistochemical assay for hypoxia in squamous cell carcinomas. CONCLUSION: The calibrated, semiquantitative assay shows promise as an approach to simplifying the quantitation of human tumor hypoxia by immunohistochemical techniques.

Results of irradiation of infiltrative lipoma in 13 dogs.

Thirteen dogs with infiltrative lipomas were treated with cobalt 60 radiation. Four of the thirteen dogs also received either whole body (n = 2) or combination local/whole body (n = 2) hyperthermia in conjunction with radiation therapy. Cytoreductive surgery was performed prior to radiation in 10 dogs, although only 3 dogs had microscopic disease at the time of radiation therapy. Dogs received a total dose of 45.6 Gy-63 Gy in 2.5-4 Gy/fraction on either a Monday/Wednesday/Friday schedule or on a daily Monday through Friday schedule. Twelve of the 13 dogs had computed tomography (CT) images acquired prior to irradiation. Survival time was determined from the time of completion of radiation therapy. Survival ranged from 6 months to 94 months, with a median (95% confidence interval) of 40 (18.5-77) months and a mean of 46.4 months. Only one dog was euthanized due to persistent signs related to the infiltrative lipoma at 6 months after the end of radiation therapy. There was no apparent difference in response based on whether or not the dogs received hyperthermia in conjunction with irradiation, although the numbers were too small to make any significant conclusions. It appears that dogs with infiltrative lipomas can benefit from external beam irradiation alone or in combination with surgery to effect long-term local tumor control.

Dynamic CT measurement of contrast medium washin kinetics in canine nasal tumors.

Tumor oxygenation affects the biologic behavior of a tumor and also its radiation response. Decreased tumor oxygenation has been associated with an aggressive phenotype and with decreased local tumor control following irradiation. Thus, measurement of oxygenation may be useful for pretreatment evaluation of a tumor. Many methods for assessing tumor oxygenation are available but most are invasive. There is a need for a non-invasive measure of oxygenation, or a surrogate for oxygenation. Measurement of perfusion has been suggested as a substitute for measurement of oxygenation. The use of washin kinetics of iodinated contrast medium to estimate perfusion has been shown to be related to radiation response of human carcinomas. We quantified the washin kinetics of iodinated contrast medium using dynamic CT in 9 dogs. All dogs had a malignant nasal tumor and perfusion was quantified at two sites in each tumor to evaluate intratumoral variation in perfusion. Dogs were given an intravenous bolus injection of contrast medium and arterial and tumor washin kinetics quantified using a helical CT scanner. Perfusion was estimated from these data using previously validated methods. Eight of the 9 dogs received definitive radiation therapy and perfusion was quantified a second time in these 8 dogs midway through irradiation. Pretreatment perfusion varied between dogs by a factor of 16.9. Between dog variation in perfusion was subjectively greater than within tumor variation based on comparison of two intratumoral regions. Changes in perfusion in individual dogs during irradiation were observed, but no identifiable pattern of perfusion alteration was detected. Measurement of perfusion in canine nasal tumors using dynamic CT is possible and further study of this parameter as it relates to radiation response is reasonable.

The computed tomographic appearance of acute thoracolumbar intervertebral disc herniations in dogs.

The appearance of herniated intervertebral disc material in the thoracolumbar vertebral canal was evaluated in 23 dogs using computed tomography (CT). The images were then compared with the myelographic and surgical findings. The normal spinal cord, outlined by epidural fat over intervertebral disc spaces, was of intermediate attenuation on transverse CT images. Herniated disc material was identified in all animals as a heterogeneous hyperattenuating extradural mass. The attenuation of the disc material increased with the degree of mineralization. In seven dogs, the herniated material was only slightly more attenuating than the spinal cord. In these dogs, small fragments of mineralized disc material and significant hemorrhage were found in the epidural space at surgery. In dogs with a long standing history of disc herniations, disc material identified in the vertebral canal had a more hyperattenuating and homogeneous appearance than recently herniated disc material. We conclude that mineralized, herniated disc material and hemorrhage can be identified quickly and safely in dogs using CT.

Hyperthermia increases accumulation of technetium-99m-labeled liposomes in feline sarcomas.

The effect of hyperthermia on the accumulation of technetium-99m-labeled liposomes was studied in feline sarcomas. Each cat received two separate injections of liposomes. The first was used to quantify the amount of technetium-99m-labeled liposomes within the tumor under normothermic conditions. The second injection was made at the beginning of a 60-min hyperthermia procedure. Planar scintigraphy was used to measure the activity of technetium-99m-labeled liposomes within the tumor at predetermined times up to 18 h after injection. Regions of interest were drawn for the tumor, lungs, liver, kidney, and aorta. Counts in the regions of interest were decay corrected. Counts/pixel in the tumor under normothermic and hyperthermic conditions were normalized to aorta counts/pixel. A total of 16 cats were eligible for the study. In two of the 16 cats, incomplete count data precluded analysis. In the remaining 14 cats, hyperthermia resulted in a significant increase in liposome accumulation in the tumor (P = 0.001). Tumor volume ranged from 1.2 to 236.2 cm3, and thermal dose ranged from 2.0 to 243.3 CEM43CT90 (equivalent time that the 10th percentile temperature was equal to 43 degrees C). There was not a relationship between either tumor volume or hyperthermia dose on the magnitude of increased liposome accumulation, suggesting that this method has application across a range of tumor volumes and degrees of heatibility.

Using units of CEM 43 degrees C T90, local hyperthermia thermal dose can be delivered as prescribed.

A randomized study was designed in dogs with spontaneous soft tissue sarcomas to gain information about the relationship between hyperthermia dose and outcome. The study compared two levels of thermal dose applied to dogs with heatable tumours, so it was necessary to deliver either a low (2-5 CEM 43 degrees C T90) or high (20-50 CEM 43 degrees C T90) thermal dose as precisely as possible. It was also desirable to have similar numbers of hyperthermia treatments in each thermal dose group. Identification of heatable tumours and randomization to high or low heat dose group was done during the first hyperthermia treatment. This was readily accomplished using mapping of temperatures in thermometry catheters, manual recording of thermal data, and visual inspection of raw thermal data with subsequent adjustment of the duration of the hyperthermia treatment. An analysis of precision of thermal dose delivery was conducted after approximately 50% of projected accrual had been met in a randomized phase III assessment of thermal dose effect. Fifty-four dogs were eligible for randomization; in 48 dogs the tumour was deemed heatable according to predetermined temperature criteria applied during the first heat treatment. Twenty-four dogs were randomized to the high heat dose group, and 24 to the low heat dose group. Median (range) total thermal dose for dogs in the high dose group was 43.5 CEM 43 degrees C T90 (16.4-66.6) compared to 3.2 CEM 43 degrees C T90 (2.1-4.6) for dogs in the low dose group. There was no overlap of thermal doses between groups. Thus, thermal dose could be delivered accurately, being within the predetermined range in 47 of the 48 dogs. Thermal dose quantified as CEM 43 degrees C T50, however, did overlap between groups and the clinical significance of this finding will not be known until outcome data are analysed. Most dogs in both groups received five hyperthermia treatments. Median (range) treatment duration for dogs in the high dose group was 300 min (147-692) compared to III min (51-381) for dogs in the low dose group. Relatively simple but accurate methods of delivering prescribed thermal dose as described herein will aid the translation of clinical hyperthermia from the research setting into more general practice once the characteristics of the relationship between hyperthermia dose and outcome are understood.

Primary irradiation of canine intracranial masses.

Twenty-nine dogs received primary radiation therapy for intracranial lesions and clinical signs suggestive of neoplasia. Presumptive diagnosis and tumor categorization was based on computed tomographic or magnetic resonance images. Meningioma was the most likely tumor type in 22 dogs and glioma or choroid plexus tumors were tentatively identified in 4 and 3 dogs, respectively. Cobalt-60 radiation was delivered in 3 Gy fractions on a daily, Monday-through-Friday basis for a total dose of 48 Gy (16 fractions) in 28 dogs; one dog received 54 Gy. Two of 29 dogs died during treatment of signs suggestive of progressive tumor growth but were included in the overall evaluation of response to treatment. Median overall survival was 250 days (range 21-804). Mild acute radiation effects on normal tissue developed and did not influence outcome in any dog. Late radiation effects could not be evaluated in this study. No significant predictive indicators were identified from the clinical or imaging data. Radiation therapy is superior to medical treatment of brain tumors in dogs with steroids, is useful for tumors that are not currently operable and may be preferable to surgical resection in dogs if the mass appears infiltrative. However, 22/29 (76%) dogs died of recurrent progressive neuropathy suggestive of tumor regrowth or progression. Thus, alternative methods for delivery of radiation to dogs with brain tumors or novel combinations of therapy should continue to undergo evaluation.

The relationship between intracellular and extracellular pH in spontaneous canine tumors.

Recently, it has been suggested that the cellular uptake of chemotherapeutic drugs may be dependent on the pH gradient between the intracellular (pHi) and extracellular (pHe) compartments. It has been demonstrated in murine tumor models that the extracellular environment is acidic, relative to the intracellular environment, thus favoring preferential accumulation of drugs that are weak acids into cells. However, concomitant measurements of pHi and pHe in spontaneous tumors have not been reported, so it is not certain how well the murine results translate to the clinical scenario. In this study, both types of measurements were performed in dogs with spontaneous malignant soft tissue tumors. On average, pHe was more acidic than pHi, with maintenance of a more physiologically balanced intracellular tumor environment. However, the magnitude of the gradient varied widely, and individual tumors had both positive and negative pH gradients (pHi - pHe). These data suggest that the magnitude and direction of the pH gradient may need to be measured for individual patient tumors and/or that manipulation of pHe may be required if exploitation of the pH gradient is to be achieved for tumor-selective augmentation of intracellular drug delivery.

The role of liposomes in drug delivery and diagnostic imaging: a review.

This manuscript is not intended as a comprehensive overview of the large filed of liposome technology and all its applications. However, our intent was to present current data, which are active, cutting-edge research. Because of their unique properties liposomes will continue to be investigated in drug delivery and imaging systems, and very likely will be incorporated into our discipline of veterinary medicine as the clinical applications of liposomes continue to expand.

Temperature-dependent changes in physiologic parameters of spontaneous canine soft tissue sarcomas after combined radiotherapy and hyperthermia treatment.

PURPOSE: The objectives of this study were to evaluate effects of hyperthermia on tumor oxygenation, extracellular pH (pHe), and blood flow in 13 dogs with spontaneous soft tissue sarcomas prior to and after local hyperthermia. METHODS AND MATERIALS: Tumor pO2 was measured using an Eppendorf polarographic device, pHe using interstitial electrodes, and blood flow using contrast-enhanced magnetic resonance imaging (MRI). RESULTS: There was an overall improvement in tumor oxygenation observed as an increase in median pO2 and decrease in hypoxic fraction (% of pO2 measurements <5 mm Hg) at 24-h post hyperthermia. These changes were most pronounced when the median temperature (T50) during hyperthermia treatment was less than 44 degrees C. Tumors with T50 > 44 degrees C were characterized by a decrease in median PO2 and an increase in hypoxic fraction. Similar thermal dose-related changes were observed in tumor perfusion. Perfusion was significantly higher after hyperthermia. Increases in perfusion were most evident in tumors with T50 < 44 degrees C. With T50 > 44 degrees C, there was no change in perfusion after hyperthermia. On average, pHe values declined in all animals after hyperthermia, with the greatest reduction seen for larger T50 values. CONCLUSION: This study suggests that hyperthermia has biphasic effects on tumor physiologic parameters. Lower temperatures tend to favor improved perfusion and oxygenation, whereas higher temperatures are more likely to cause vascular damage, thus leading to greater hypoxia. While it has long been recognized that such effects occur in rodent tumors, this is the first report to tie such changes to temperatures achieved during hyperthermia in the clinical setting. Furthermore, it suggests that the thermal threshold for vascular damage is higher in spontaneous tumors than in more rapidly growing rodent tumors.

Acute pancreatitis associated with administration of a nitric oxide synthase inhibitor in tumor-bearing dogs.

BACKGROUND: Nitric oxide synthase (NOS) inhibitors have been investigated as potential cytotoxic agents to treat tumors lacking p53 function. Furthermore, their ability to reduce tumor blood flow can be combined with drugs that are specifically designed to kill cells that are hypoxic or to improve temperatures during local heat (hyperthermia) treatment of tumors. This paper reports the unexpected development of acute pancreatitis in two tumor-bearing pet dogs that were treated with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) during administration of local hyperthermia. METHODS: Prior to the use of L-NAME in tumor-bearing dogs, purpose-bred beagles were studied. Following induction of inhalation anesthesia, local hyperthermia was applied to either normal thigh muscle (beagles) or tumors (tumor-bearing dogs). Once a thermal steady state was achieved, L-NAME was administered and temperature monitoring continued. Animals were observed after treatment for evidence of toxicity. RESULTS: The beagles tolerated the treatment well, with no side effects noted either clinically or by routine CBC or blood chemistry analyses. In contrast, the first two tumor-bearing dogs accrued onto the phase I study developed acute pancreatitis in the immediate post-treatment period which necessitated hospitalization and intensive care. The trial was stopped. Both dogs had intercurrent risk factors which predisposed them to development of pancreatitis, although neither had a history of symptoms of pancreatitis at the time the hyperthermia + L-NAME treatment was given. CONCLUSIONS: We conclude that caution should be exercised when considering NOS inhibition for cancer treatment. Careful evaluation of history and health status as well as recognition of potential risk factors may be key in avoiding potentially fatal complications. This study demonstrates the value of performing potentially harmful treatments in tumor-bearing dogs prior to introduction into the human clinic.