Katherine S. Hansen

High frequency of BRAF mutations in nevi.

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

CCNU in the Treatment of Canine Epitheliotropic Lymphoma

This retrospective study examined the use of CCNU (1-[2-chloroethyl]3-cyclohexyl-1-nitrosurea) in 36 dogs with epitheliotropic lymphoma. Thirty-one (86%) dogs had the cutaneous form of disease, and 5 (14%) dogs had the oral form of disease. Nineteen (51%) dogs were treated with other chemotherapeutic agents before receiving CCNU. All dogs had detectable disease at the time CCNU therapy was initiated. Dogs received a median starting CCNU dosage of 70 mg/m2 (range, 50-100 mg/m2). The median number of treatments administered was 3 (range, 1-12 treatments). After the initial treatment, the CCNU dosage was adjusted in 9 of 26 (35%) dogs in which CCNU was continued: 7 had dosage reductions, and 2 had dosage escalations. Twenty-eight of 36 (78%) dogs had a measurable response to CCNU for a median duration of 106 days (95% confidence interval [CI], 75-182). Six dogs (17%) had a complete response, including 5 dogs with the cutaneous form and 1 dog with the oral form. Twenty-two dogs (61%) had a partial response, including 20 dogs with the cutaneous form and 2 dogs with the oral form, for a median duration of 88 days (95% CI, 62-170). Toxicoses after CCNU chemotherapy included myelosuppression in up to 29% of the dogs, gastrointestinal signs in up to 22% of the dogs, and liver enzyme activity increases in up to 86% of the dogs. This study demonstrates that CCNU chemotherapy can be considered a reasonable option for the treatment of canine epitheliotropic lymphoma in dogs.

Delivery rate affects uptake of a fluorescent glucose analog in murine metastatic breast cancer.

We demonstrate an optical strategy using intravital microscopy of dorsal skin flap window chamber models to image glucose uptake and vascular oxygenation in vivo. Glucose uptake was imaged using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). SO2 was imaged using the differential absorption properties of oxygenated [HbO2] and deoxygenated hemoglobin [dHb]. This study was carried out on two sibling murine mammary adenocarcinoma lines, 4T1 and 4T07. 2-NBDG uptake in the 4T1 tumors was lowest when rates of delivery and clearance were lowest, indicating perfusion-limited uptake in poorly oxygenated tumor regions. For increasing rates of delivery that were still lower than the glucose consumption rate (as measured in vitro), both 2-NBDG uptake and the clearance rate from the tumor increased. When the rate of delivery of 2-NBDG exceeded the glucose consumption rate, 2-NBDG uptake decreased with any further increase in rate of delivery, but the clearance rate continued to increase. This inflection point was not observed in the 4T07 tumors due to an absence of low delivery rates close to the glucose consumption rate. In the 4T07 tumors, 2-NBDG uptake increased with increasing rates of delivery at low rates of clearance. Our results demonstrate that 2-NBDG uptake in tumors is influenced by the rates of delivery and clearance of the tracer. The rates of delivery and clearance are, in turn, dependent on vascular oxygenation of the tumors. Knowledge of the kinetics of tracer uptake as well as vascular oxygenation is essential to make an informed assessment of glucose demand of a tumor.

PET with 62Cu-ATSM and 62Cu-PTSM is a useful imaging tool for hypoxia and perfusion in pulmonary lesions.

OBJECTIVE Hypoxia is a characteristic of many tumors and portends a worse prognosis in lung, cervical, prostate, and rectal cancers. Unlike the others, lung cancers present a unique challenge in measuring hypoxia, with invasive biopsies and higher rates of complications. Noninvasive imaging studies detecting hypoxia using isotopes of copper-diacetyl-bis(N4-methylthiosemicarbazone) ((62)Cu-ATSM) have predicted prognosis and treatment outcomes in some small feasibility trials. These images, however, may not identify all areas of hypoxia. Hence, we hypothesize that the addition of another PET imaging agent, copper-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((62)Cu-PTSM), which can detect areas of perfusion, can augment the information obtained in (62)Cu-ATSM PET scans. SUBJECTS AND METHODS To characterize tumors on the basis of both perfusion and hypoxia, 10 patients were studied using both (62)Cu-ATSM and (62)Cu-PTSM PET scans. In addition, proteomic arrays looking at specific proangiogenic, survival, and proinflammatory targets were assessed. RESULTS Six of 10 patients had evaluable PET scans. Our initial experience of characterizing lung tumor hypoxia using (62)Cu-ATSM and (62)Cu-PTSM PET scans showed that visualization of areas with hypoxia normalized for perfusion is feasible. All studied tumors exhibited some hypoxia. Despite the small sample size, a positive relationship was noted between epidermal growth factor levels and (62)Cu-ATSM-detected hypoxia. CONCLUSION This initial series of (62)Cu-ATSM and (62)Cu-PTSM PET scans shows that evaluating lung masses by visualizing hypoxia and perfusion is a feasible and novel technique to provide more information. Further investigation is warranted to assess the potential role of (62)Cu-ATSM and (62)Cu-PTSM PET techniques combined with proteomics as alternatives to invasive biopsy techniques in clinical care.

VALIDATION OF AN INDEXED RADIOTHERAPY HEAD POSITIONING DEVICE FOR USE IN DOGS AND CATS.

Setup variability affects the appropriate delivery of radiation and informs the setup margin required to treat radiation patients. Twenty-four veterinary patients with head and neck cancers were enrolled in this prospective, cross-sectional study to determine the accuracy of an indexed board immobilization device for positioning. Couch position values were defined at the first treatment based on setup films. At subsequent treatments, patients were moved to the previously defined couch location, orthogonal films were acquired, table position was modified, and displacement was recorded. The mean systematic displacement, random displacement, overall displacement, and mean displacement values of the three-dimensional (3D) vector were calculated. Three hundred thirty-two pairs of orthogonal setup films were analyzed for displacement in cranial-caudal, lateral, and dorsal-ventral directions. The mean systematic displacements were 0.5, 0.8, and 0.5 mm, respectively. The mean random displacements were 1.0, 1.1, and 0.7 mm, respectively. The overall displacements were 1.1, 1.4, and 0.9 mm, respectively. The mean 3D vector value was 1.6 mm with a standard deviation of 1.2 mm. Ninety-five percent of the vectors were <3.6 mm. These values were compared to data obtained with a previously used immobilization device. A t-test was used to compare the two devices. The 3D vector, random displacement in all directions, and overall displacement in the cranial-caudal and dorsal-ventral directions were significantly smaller than displacements with the previous device. The precision and accuracy of the indexed board device was superior to the historical head and neck device.

INTER- AND INTRAFRACTION MOTION FOR STEREOTACTIC RADIOSURGERY IN DOGS AND CATS USING A MODIFIED BRAINLAB FRAMELESS STEREOTACTIC MASK SYSTEM.

Precise and accurate patient positioning is necessary when doing stereotactic radiosurgery (SRS) to ensure adequate dosing to the tumor and sparing of normal tissues. This prospective cross-sectional study aimed to assess feasibility of a commercially available modified frameless SRS positioning system for use in veterinary radiotherapy patients with brain tumors. Fifty-one dogs and 12 cats were enrolled. Baseline and verification CT images were acquired. The verification CT images from 32 dogs and five cats had sufficient images for fusion to baseline CT images. A rigid box-based fusion was performed to determine interfraction motion. Forty-eight dogs and 11 cats were assessed for intrafraction motion by cine CT. Seventy percent of dogs and 60% of cats had interfraction 3D vector translational shifts >1 mm, with mean values of 1.9 mm in dogs, and 1.8 mm in cats. In dogs muscle wasting was weakly correlated with translational shifts. The maximum angular interfraction motion observed was 6.3° (roll), 3.5° (pitch), and 3.3° (yaw). There was no correlation between angular interfraction motion and weight, brachycephaly, or muscle wasting. Fifty-seven percent of dogs and 50% of cats had respiration-related intrafraction motion. Of these, 4.5% of dogs and 10% of cats had intrafraction motion >1 mm. This study demonstrates the modified Brainlab system is feasible for SRS in dogs and cats. The smaller cranial size and difference in anatomy increases setup uncertainty in some animals beyond limits usually accepted in SRS. Image-guided positioning is recommended to achieve clinically acceptable setup accuracy (<1 mm) for SRS.

Can a commercially available EPID dosimetry system detect small daily patient setup errors for cranial IMRT/SRS?

PURPOSE The purpose of this study was to determine if the Sun Nuclear PerFRACTION electronic portal imager device dosimetry software would be able to detect setup errors in a clinical setting and would be able to correctly identify the direction in which the setup error was introduced. METHODS AND MATERIALS A 7-field intensity modulated radiation therapy (IMRT) treatment plan for a centrally located tumor was developed for 1 phantom and 5 canine cadaver heads. Systematic setup errors were introduced by manually moving the treatment couch by 1, 3, and 5 mm in each translational direction to assess stereotactic radiation surgery (SRS), IMRT, and 3-dimensional (3D) treatment tolerances after the initial alignment was performed. An angular setup error of 5° yaw was also assessed. The delivered treatment fluence was automatically imported in the PerFRACTION software and compared with the baseline fluence. RESULTS In the canine phantom, a 5-mm shift was undetected by gamma analysis, and up to a 2-cm shift had to be introduced for the gamma pass rate of 3%/3 mm to fall below a 95% pass rate criterion. The same 5-mm shift using 3% difference caused the pass rates for 2 fields to drop below the 95% tolerance. For each respective translational shift, the affected beam angles were consistent across the cadaver heads and correlated with the direction of translational shift. The best field pass rate, worst field pass rate, and average pass rate across all 7 fields was analyzed to develop clinical guidance on parameter settings for SRS, IMRT, and 3D tolerances. CONCLUSIONS PerFRACTION 2-dimensional mode successfully detected setup errors outside the systematic error tolerance for SRS, IMRT, and 3D when an appropriate analysis metric and pass/fail criteria was implemented. Our data confirm that percent difference may be more sensitive in detecting plan failure than gamma analysis.

Treatment of MRI-Diagnosed Trigeminal Peripheral Nerve Sheath Tumors by Stereotactic Radiotherapy in Dogs.

Background Stereotactic radiotherapy (SRT) is an emerging technique for treating tumors in animals. Objectives To assess the outcome of dogs with suspected intracranial trigeminal nerve peripheral nerve sheath tumors (PNST) treated with SRT. Animals Eight dogs with presumptive PNST. Methods This was a retrospective study of dogs identified by searching UC Davis Veterinary Medical Teaching Hospital medical records for dogs treated with SRT for a presumed PNST. Presumptive diagnosis was based on magnetic resonance imaging. SRT was delivered in 3 dose fractions of 8 Gray (Gy) on consecutive days or every other day to a total dose of 24 Gy. Results Median disease‐specific survival was 745 days (range: 99–1375 days, n = 6). No signs of acute adverse effects of radiation treatment were recorded. Late radiation effects versus tumor progression could not be confirmed histopathologically because of few animals undergoing necropsy. Conclusions and Clinical Importance This study provides preliminary evidence that dogs with PNST benefit from SRT in terms of long‐term survival. The treatment appears to be well tolerated and requires fewer anesthetic events for animals compared to full‐course radiation.

A novel, removable, cerrobend, beam-blocking device for radiation therapy of the canine head and neck: Pilot study

Radiation therapy of the head and neck can result in mucositis and other acute affects in the oral cavity. This prospective pilot study evaluated a novel, intraoral, beam-blocking device for use during imaging and therapeutic procedures. The beam-blocking device was made from a metal alloy inserted into a coated frozen dessert mold (Popsicle® Mold, Cost Plus World Market, Oakland, CA). The device was designed so that it could be inserted into an outer shell, which in turn allowed it to be placed or removed depending on the need due to beam configuration. A Farmer type ionization chamber and virtual water phantom were used to assess effects of field size on transmission. Six large breed cadaver dogs, donated by the owner after death, were recruited for the study. Delivered dose at the dorsal and ventral surfaces of the device, with and without the alloy block in place, were measured using radiochromic film. It was determined that transmission was field size dependent with larger field sizes leading to decreased attenuation of the beam, likely secondary to scatter. The mean and median transmission on the ventral surface without the beam-blocking device was 0.94 [range 0.94-0.96]. The mean and median transmission with the beam-blocking device was 0.52 [range 0.50-0.57]. The mean and median increase in dose due to backscatter on the dorsal surface of the beam-blocking device was 0.04 [range 0.02-0.04]. Findings indicated that this novel device can help attenuate radiation dose ventral to the block in dogs, with minimal backscatter.

Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.