Pretargeted PET of Osteodestructive Lesions in Dogs.

The last decade has witnessed the creation of a highly effective approach to in vivo pretargeting based on the inverse electron demand Diels-Alder (IEDDA) click ligation between tetrazine (Tz) and trans-cyclooctene (TCO). Despite the steady progression of this technology toward the clinic, concerns have persisted regarding whether this in vivo chemistry will work in humans given their larger size and blood volume. In this work, we describe the use of a 64Cu-labeled Tz radioligand ([64Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted positron emission tomography (PET) imaging of osteodestructive lesions in a large animal model: companion dogs. First, in a small animal pilot study, healthy mice were injected with TCO-BP followed after 1 or 6 h by [64Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [64Cu]Cu-SarAr-Tz, revealing that this approach produced high activity concentrations in the bone (>20 and >15%ID/g in the femur and humerus, respectively, at 24 h post injection) as well as high target-to-background contrast. Subsequently, companion dogs (n = 5) presenting with osteodestructive lesions were administered TCO-BP (5 or 10 mg/kg) followed 1 h later by [64Cu]Cu-SarAr-Tz (2.2-7.3 mCi; 81.4-270.1 MBq). PET scans were collected for each dog 4 h after the administration of the radioligand, and SUV values for the osteodestructive lesions, healthy bones, and kidneys were determined. In these animals, pretargeted PET clearly delineated healthy bone and produced very high activity concentrations in osteodestructive lesions. Low levels of uptake were observed in all healthy organs except for the kidneys and bladder due to the renal excretion of excess radioligand. Ultimately, this work not only illustrates that pretargeted PET with TCO-BP and [64Cu]Cu-SarAr-Tz is an effective tool for the visualization of osteodestructive lesions but also demonstrates for the first time that in vivo pretargeting based on IEDDA click chemistry is feasible in large animals.

Biodistribution and image characteristics of 124 I-positron emission tomography in dogs with neuroendocrine neoplasia.

Radioactive iodine is frequently used for staging of human thyroid carcinomas. Iodine-124 scans performed using position emission tomography (PET) allow for more precise dosimetry of therapeutic radioiodine. The distribution of I-124 has not previously been described in veterinary medicine. The purpose of this prospective, exporatory, descriptive study is to evaluate the whole-body distribution of I-124 in dogs with suspected thyroid carcinoma. Ten dogs with either a cytologic diagnosis of a neuroendocrine neoplasm or biochemical hyperthyroidism were enrolled in a prospective clinical study. Whole-body I-124 PET/CT scans were performed and were evaluated for physiologic and pathologic uptake of I-124. The maximum and mean standardized uptake values (SUVmean) were recorded for several normal and abnormal tissues. Varying degrees of uptake were found in thyroid tumors (SUVmean = 66.37), ectopic thyroid masses (21.44), presumed metastatic lesions in lymph nodes (32.14), and the pulmonary parenchyma (4.50). In most dogs, physiologic uptake above background, measured in maximum SUV, was identified in parotid and mandibular salivary glands (14.00 and 1.57) the urinary tract (1.83), the gastrointestinal tract (19.90 stomach, 6.15 colon), the liver (1.41), and the heart (1.88). Occasionally, uptake was identified in the nasolacrimal duct (3.42), salivary duct (2.73), gallbladder (2.68), and anal gland (2.22). Physiologic uptake was also identified in normal thyroid glands and ectopic thyroid tissue. This study provides a baseline of pathologic and physiologic uptake of I-124 in dogs with thyroid carcinoma, to guide interpretation of future studies.

Immune cell ß2-adrenergic receptors contribute to the development of heart failure.

ß-Adrenergic receptors (ßARs) regulate normal and pathophysiological heart function through their impact on contractility. ßARs are also regulators of immune function where they play a unique role depending on the disease condition and immune cell type. Emerging evidence suggests an important role for the ß2AR subtype in regulating remodeling in the pathological heart; however, the importance of these responses has never been examined. In heart failure, catecholamines are elevated, leading to chronic ßAR activation and contributing to the detrimental effects in the heart. We hypothesized that immune cell ß2AR plays a critical role in the development of heart failure in response to chronic catecholamine elevations through their regulation of immune cell infiltration. To test this, chimeric mice were generated by performing bone marrow transplant (BMT) experiments using wild-type (WT) or ß2AR knockout (KO) donors. WT and ß2ARKO BMT mice were chronically administered the ßAR agonist isoproterenol. Immune cell recruitment to the heart was examined by histology and flow cytometry. Numerous changes in immune cell recruitment were observed with isoproterenol administration in WT BMT mice including proinflammatory myeloid populations and lymphocytes with macrophages made up the majority of immune cells in the heart and which were absent in ß2ARKO BMT animal. ß2ARKO BMT mice had decreased cardiomyocyte death, hypertrophy, and interstitial fibrosis following isoproterenol treatment, culminating in improved function. These findings demonstrate an important role for immune cell ß2AR expression in the heart's response to chronically elevated catecholamines.NEW & NOTEWORTHY Immune cell ß2-adrenergic receptors (ß2ARs) are important for proinflammatory macrophage infiltration to the heart in a chronic isoproterenol administration model of heart failure. Mice lacking immune cell ß2AR have decreased immune cell infiltration to their heart, primarily proinflammatory macrophage populations. This decrease culminated to decreased cardiac injury with lessened cardiomyocyte death, decreased interstitial fibrosis and hypertrophy, and improved function demonstrating that ß2AR regulation of immune responses plays an important role in the heart's response to persistent ßAR stimulation.

ß2-Adrenergic Receptors Increase Cardiac Fibroblast Proliferation Through the Gαs/ERK1/2-Dependent Secretion of Interleukin-6.

Fibroblasts are an important resident cell population in the heart involved in maintaining homeostasis and structure during normal conditions. They are also crucial in disease states for sensing signals and initiating the appropriate repair responses to maintain the structural integrity of the heart. This sentinel role of cardiac fibroblasts occurs, in part, through their ability to secrete cytokines. ß-adrenergic receptors (ßAR) are also critical regulators of cardiac function in the normal and diseased state and a major therapeutic target clinically. ßAR are known to influence cytokine secretion in various cell types and they have been shown to be involved in cytokine production in the heart, but their role in regulating cytokine production in cardiac fibroblasts is not well understood. Thus, we hypothesized that ßAR activation on cardiac fibroblasts modulates cytokine production to influence fibroblast function. Using primary fibroblast cultures from neonatal rats and adult mice, increased interleukin (IL)-6 expression and secretion occurred following ß2AR activation. The use of pharmacological inhibitors and genetic manipulations showed that IL-6 elevations occurred through the Gαs-mediated activation of ERK1/2 and resulted in increased fibroblast proliferation. In vivo, a lack of ß2AR resulted in increased infarct size following myocardial infarction and impaired wound closure in a murine dermal wound healing assay. These findings identify an important role for ß2AR in regulating fibroblast proliferation through Gαs/ERK1/2-dependent alterations in IL-6 and may lead to the development of improved heart failure therapies through targeting fibrotic function of ß2AR.

Therapeutic efficacy of boron neutron capture therapy mediated by boron-rich liposomes for oral cancer in the hamster cheek pouch model.

The application of boron neutron capture therapy (BNCT) mediated by liposomes containing (10)B-enriched polyhedral borane and carborane derivatives for the treatment of head and neck cancer in the hamster cheek pouch oral cancer model is presented. These liposomes are composed of an equimolar ratio of cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine, incorporating K[nido-7-CH3(CH2)15-7,8-C2B9H11] (MAC) in the bilayer membrane while encapsulating the hydrophilic species Na3[ae-B20H17NH3] (TAC) in the aqueous core. Unilamellar liposomes with a mean diameter of 83 nm were administered i.v. in hamsters. After 48 h, the boron concentration in tumors was 67 ± 16 ppm whereas the precancerous tissue contained 11 ± 6 ppm, and the tumor/normal pouch tissue boron concentration ratio was 10:1. Neutron irradiation giving a 5-Gy dose to precancerous tissue (corresponding to 21 Gy in tumor) resulted in an overall tumor response (OR) of 70% after a 4-wk posttreatment period. In contrast, the beam-only protocol gave an OR rate of only 28%. Once-repeated BNCT treatment with readministration of liposomes at an interval of 4, 6, or 8 wk resulted in OR rates of 70-88%, of which the complete response ranged from 37% to 52%. Because of the good therapeutic outcome, it was possible to extend the follow-up of BNCT treatment groups to 16 wk after the first treatment. No radiotoxicity to normal tissue was observed. A salient advantage of these liposomes was that only mild mucositis was observed in dose-limiting precancerous tissue with a sustained tumor response of 70-88%.

Use of an electronic brachytherapy surface applicator to treat an epiglottal fibrosarcoma in a dog.

Presented is the case of an epiglottal fibrosarcoma in a dog. The location of the mass resulted in challenges in the delivery of adequate dose to the tumor, and herein we describe the treatment using an electronic brachytherapy source. The treatment consisted of four Gy fractions, twice daily for a total of 10 fractions (40 Gy total). Visual reevaluation two weeks after treatment supported adequate spatial dose delivery, and the patient was reportedly improved six weeks after treatment. We demonstrate that plesiotherapy using an electronic brachytherapy device is feasible and may be useful in the treatment of carefully selected veterinary tumors.

Boron neutron capture therapy demonstrated in mice bearing EMT6 tumors following selective delivery of boron by rationally designed liposomes.

The application of boron neutron capture therapy (BNCT) following liposomal delivery of a (10)B-enriched polyhedral borane and a carborane against mouse mammary adenocarcinoma solid tumors was investigated. Unilamellar liposomes with a mean diameter of 134 nm or less, composed of an equimolar mixture of cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine and incorporating Na3[1-(2'-B10H9)-2-NH3B10H8] in the aqueous interior and K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the bilayer, were injected into the tail veins of female BALB/c mice bearing right flank EMT6 tumors. Biodistribution studies indicated that two identical injections given 24 h apart resulted in tumor boron levels exceeding 67 µg/g tumor at 54 h--with tumor/blood boron ratios being greatest at 96 h (5.68:1; 43 µg boron/g tumor)--following the initial injection. For BNCT experiments, tumor-bearing mice were irradiated 54 h after the initial injection for 30 min with thermal neutrons, resulting in a total fluence of 1.6 × 10(12) neutrons per cm(2) (±7%). Significant suppression of tumor growth was observed in mice given BNCT vs. control mice (only 424% increase in tumor volume at 14 d post irradiation vs. 1551% in untreated controls). In a separate experiment in which mice were given a second injection/irradiation treatment 7 d after the first, the tumor growth was vastly diminished (186% tumor volume increase at 14 d). A similar response was obtained for mice irradiated for 60 min (169% increase at 14 d), suggesting that neutron fluence was the limiting factor controlling BNCT efficacy in this study.

Translational history and hope of immunotherapy of canine tumors.

Companion dogs have served an important role in cancer immunotherapy research. Sharing similar environments and diets with humans, dogs naturally develop many of the same cancers. These shared exposures, coupled with dogs' diverse genetic makeup, makes them ideal subjects for studying cancer therapies. Tumors like osteosarcoma (cOSA), hemangiosarcoma (cHSA), soft-tissue sarcoma (cSTS), and non-Hodgkin lymphoma (cnHL) occur with greater frequency than their counterpart disease in humans. Canine brain tumors allow study of therapy strategies with imaging, surgery, and radiotherapy equipment in veterinary patients with near-human geometry. Non-specific immunostimulants, autologous and allogeneic vaccines, immune checkpoint inhibitors, and cellular therapies used treating canine cancers have been tested in veterinary clinical trials. These treatments have not only improved outcomes for dogs but have also provided valuable insights for human cancer treatment. Advancements in radiation technology and the development of tools to characterize canine immune responses have further facilitated the ability to translate veterinary clinical trial results to human applications. Advancements in immunotherapy of canine tumors have directly supported translation to human clinical trials leading to approved therapies for cancer patients around the world. The study of immunotherapy in dogs has been and will continue to be a promising avenue for advancing human cancer treatment.

The role of companion animal models in radiopharmaceutical development and translation.

Advancements in molecular imaging and drug targeting have created a renaissance in the development of radiopharmaceuticals for therapy and theranostics. While some radiopharmaceuticals, such as Na[131I]I, have been used clinically for decades, new agents are being approved using small-molecules, peptides, and antibodies for targeting. As these agents are being developed, the need to understand dosimetry and biologic effects of the systemically delivered radiotherapy becomes more important, particularly as highly potent radiopharmaceuticals using targeted alpha therapy become clinically utilized. As the processes being targeted become more complex, and the radiobiology of different particulate radiation becomes more diverse, models that better recapitulate human cancer and geometry are necessary. Companion animals develop many of the same types of cancer, carrying many of the same genetic drivers as those seen in people, and the scale and geometry of tumours in dogs more closely mimics those in humans than murine tumour models. Key translational challenges in oncology, such as alterations in tumour microenvironment, hypoxia, heterogeneity, and geometry are addressed by companion animal models. This review paper will provide background on radiopharmaceutical targeting techniques, review the use of radiopharmaceuticals in companion animal oncology, and explore the translational value of treating these patients in terms of dosimetry, treatment outcomes, and normal tissue complication rates.

Single Chelator-Minibody Theranostic Agents for 89Zr PET Imaging and 177Lu Radiopharmaceutical Therapy of PSMA-Expressing Prostate Cancer.

Here we describe an anti-prostate-specific membrane antigen (PSMA) minibody (IAB2MA) conjugated to an octadentate, macrocyclic chelator based on four 1-hydroxypyridin-2-one coordinating units (Lumi804 [L804]) labeled with 89Zr (PET imaging) and 177Lu (radiopharmaceutical therapy), with the goal of developing safer and more efficacious treatment options for prostate cancer. Methods: L804 was compared with the current gold standard chelators, DOTA and deferoxamine (DFO), conjugated to IAB2MA for radiolabeling with 177Lu and 89Zr in cell binding, preclinical biodistribution, imaging, dosimetry, and efficacy studies in the PSMA-positive PC3-PIP tumor-bearing mouse model of prostate cancer. Results: Quantitative radiolabeling (>99% radiochemical yield) of L804-IAB2MA with 177Lu or 89Zr was achieved at ambient temperature in under 30 min, comparable to 89Zr labeling of DFO-IAB2MA. In contrast, DOTA-IAB2MA was radiolabeled with 177Lu for 30 min at 37°C in approximately 90% radiochemical yield, requiring further purification. Using europium(III) as a luminescent surrogate, high binding affinity of Eu-L804-IAB2MA to PSMA was demonstrated in PC3-PIP cells (dissociation constant, 4.6 ± 0.6 nM). All 4 radiolabeled constructs showed significantly higher levels of internalization after 30 min in the PC3-PIP cells than in PSMA-negative PC3-FLU cells. The accumulation of 177Lu- and 89Zr-L804-IAB2MA in PC3-PIP tumors and all organs examined (i.e., heart, liver, spleen, kidney, muscle, salivary glands, lacrimal glands, carcass, and bone) was significantly lower than that of 177Lu-DOTA-IAB2MA and 89Zr-DFO-IAB2MA at 96 and 72 h after injection, respectively. Generally, SPECT/CT and PET/CT imaging data showed no significant difference in the SUVmean of the tumors or muscle between the radiotracers. Dosimetry analysis via both organ-level and voxel-level dose calculation methods indicated significantly higher absorbed doses of 177Lu-DOTA-IAB2MA in tumors, kidney, liver, muscle, and spleen than of 177Lu-L804-IAB2MA. PC3-PIP tumor-bearing mice treated with single doses of 177Lu-L804-IAB2MA (18.4 or 22.2 MBq) exhibited significantly prolonged survival and reduced tumor volume compared with unlabeled minibody control. No significant difference in survival was observed between groups of mice treated with 177Lu-L804-IAB2MA or 177Lu-DOTA-IAB2MA (18.4 or 22.2 MBq). Treatment with 177Lu-L804-IAB2MA resulted in lower absorbed doses in tumors and less toxicity than that of 177Lu-DOTA-IAB2MA. Conclusion: 89Zr- and 177Lu-L804-IAB2MA may be a promising theranostic pair for imaging and therapy of prostate cancer.

Monte Carlo dosimetric analyses on the use of90Y-IsoPet intratumoral therapy in canine subjects.

Objective.To investigate different dosimetric aspects of90Y-IsoPet™ intratumoral therapy in canine soft tissue sarcomas, model the spatial spread of the gel post-injection, evaluate absorbed dose to clinical target volumes, and assess dose distributions and treatment efficacy.Approach.Six canine cases treated with90Y-IsoPet™ for soft tissue sarcoma at the Veterinary Health Center, University of Missouri are analyzed in this retrospective study. The dogs received intratumoral IsoPet™ injections, following a grid pattern to achieve a near-uniform dose distribution in the clinical target volume. Two dosimetry methods were performed retrospectively using the Monte Carlo toolkit OpenTOPAS: imaging-based dosimetry obtained from post-injection PET/CT scans, and stylized phantom-based dosimetry modeled from the planned injection points to the gross tumor volume. For the latter, a Gaussian parameter with variable sigma was introduced to reflect the spatial spread of IsoPet™. The two methods were compared using dose-volume histograms (DVHs) and dose homogeneity, allowing an approximation of the closest sigma for the spatial spread of the gel post-injection. In addition, we compared Monte Carlo-based dosimetry with voxel S-value (VSV)-based dosimetry to investigate the dosimetric differences.Main results.Imaging-based dosimetry showed differences between Monte Carlo and VSV calculations in tumor high-density areas with higher self-absorption. Stylized phantom-based dosimetry indicated a more homogeneous target dose with increasing sigma. The sigma approximation of the90Y-IsoPet™ post-injection gel spread resulted in a median sigma of approximately 0.44 mm across all cases to reproduce the dose heterogeneity observed in Monte Carlo calculations.Significance.The results indicate that dose modeling based on planned injection points can serve as a first-order approximation for the delivered dose in90Y-IsoPet™ therapy for canine soft tissue sarcomas. The dosimetry evaluation highlights the non-uniformity of absorbed doses despite the gel spread, emphasizing the importance of considering tumor dose heterogeneity in treatment evaluation. Our findings suggest that using Monte Carlo for dose calculation seems more suitable for this type of tumor where high-density areas might play an important role in dosimetry.

Sodium iodide symporter immunolabelling as a predictor of clinical iodide uptake in canine thyroid carcinoma: A preliminary study.

Thyroid follicular tumours may take up iodide via the sodium-iodide symporter. Knowledge of iodide uptake could then allow treatment with I-131 in dogs with high-risk tumours. The objective of this study was to determine the relationship between clinically detectable iodide uptake (as determined by scintigraphy and/or thyroxine concentrations) and sodium iodide symporter immunohistochemical labelling on histologically fixed thyroid tumours. Nineteen dogs were identified who were diagnosed with thyroid carcinoma and underwent surgery from November 2017 to July 2021. All had recorded thyroid hormone concentrations and were hyperthyroid and/or underwent preoperative nuclear imaging using planar scintigraphy (technetium-99m or I-123), or I-124 PET-CT. All dogs subsequently underwent surgery to remove the thyroid mass. Twenty-two tumours were submitted for histopathologic analysis immediately following surgery, which confirmed a diagnosis of thyroid carcinoma for each tumour. Images and/or thyroid hormone concentrations were reviewed for the included cases, and tumours were sorted into an avid/functional group (group 1) and a non-avid/functional group (group 2). The tumour tissues were re-examined histologically using sodium iodide symporter (NIS) immunohistochemistry (IHC). Group 1 contained 15 avid/functional tumours. Twelve of these tumours had membranous NIS IHC labelling. Group 2 contained 7 non-avid tumours. One of these tumours had membranous NIS IHC labelling. This resulted in an overall sensitivity and specificity for identification of avid/functional tumours with membranous NIS of 80.0% and 85.7%, respectively. NIS IHC may predict ion trapping in canine follicular thyroid tumours. Further studies using iodide-based imaging are warranted to better determine the clinical utility of this diagnostic modality.

Effects of neoadjuvant zoledronate and radiation therapy on cell survival, cell cycle distribution, and clinical status in canine osteosarcoma.

Introduction: Zoledronic acid (ZOL) is a third-generation bisphosphonate with a higher affinity for bone resorption areas than earlier bisphosphonates (i.e., pamidronate, PAM). In human medicine, ZOL provides improved bone pain relief and prolonged time to skeletal-related events compared to its older generational counterparts. Preclinical studies have investigated its role as an anti-neoplastic agent, both independently and synergistically, with radiation therapy (RT). ZOL and RT act synergistically in several neoplastic human cell lines: prostate, breast, osteosarcoma, and fibrosarcoma. However, the exact mechanism of ZOL's radiosensitization has not been fully elucidated. Methods: We investigated ZOL's ability to induce apoptosis in canine osteosarcoma cell lines treated with various doses of megavoltage external beam radiotherapy. Second, we evaluated cell cycle arrest in ZOL-treated cells to assess several neo-adjuvant time points. Finally, we treated 20 dogs with naturally occurring appendicular OS with 0.1 mg/kg ZOL IV 24 h before receiving 8 Gy of RT (once weekly fraction x 4 weeks). Results: We found that apoptosis was increased in all ZOL-treated cell lines compared to controls, and the combination of ZOL and RT resulted in dissimilar apoptosis between Abrams and D-17 and HMPOS cell lines. Cell cycle arrest (G2/M phase) was minimal and variable between cell lines but perhaps greatest at 48 h post-ZOL treatment. Only 10% of dogs treated with ZOL and RT developed pathologic fractures, compared to 44% of dogs historically treated with PAM and RT (p = 0.027). Discussion: ZOL and RT appear to be a well-tolerated combination treatment scheme for non-surgical candidates; future studies must elucidate the ideal timing of ZOL.

Surgery and postoperative definitive radiotherapy for management of canine soft tissue sarcoma: a multi-institutional retrospective study of 272 dogs (2010-2020).

OBJECTIVE: To report local progression and survival in dogs following surgery and postoperative definitive radiotherapy (dRT) for management of soft tissue sarcoma (STS) and to evaluate risk factors for local progression and survival. METHODS: Records were retrospectively reviewed at 9 referral hospitals for dogs managed with postoperative dRT between January 1, 2010, and January 1, 2020, following surgery for STS. Data related to presentation, surgery, dRT, systemic therapy, and outcome were abstracted. Selected variables were assessed for association with local progression and overall survival. RESULTS: 272 dogs were included. Histologic grade was reported in 249 dogs: 102 were grade 1 (40.9%), 120 were grade 2 (48.2%), and 27 were grade 3 (10.8%). Local progression was suspected or confirmed in 56 dogs. Local progression rates were similar for grade 1 (24 of 89 [26.7%]), grade 2 (23 of 111 [20.7%]), and grade 3 tumors (6 of 22 [27.3%]). Previous recurrence (P = .010) and subsequent distant metastasis (P = .014) were associated with more frequent local progression; intensity-modulated radiotherapy was associated with decreased local progression (P = .025) compared to other forms of delivery. Age (P = .049), grade (P = .009), previous recurrence (P = .009), and institution type for surgery (P = .043) were associated with overall survival. CONCLUSIONS: Outcomes for most dogs were good; however, the frequency of local progression indicates an ongoing need to critically appraise local management strategies, particularly for low-grade STS. Intensity-modulated radiotherapy was associated with lower rates of local progression and may be preferred to less precise forms of delivery. CLINICAL RELEVANCE: These data may guide clinicians when making decisions regarding dRT for management of STS.

Fracture rate and time to fracture in dogs with appendicular osteosarcoma receiving finely fractionated compared to coarsely fractionated radiation therapy: A single institution study.

BACKGROUND: Radiation therapy (RT) is used for local pain alleviation in dogs with appendicular osteosarcoma (OS), especially among dogs that are poor surgical candidates for amputation. However, many historical reports of fractionated protocols lack time to fracture and fracture rates. OBJECTIVES: The primary objectives of this retrospective study were to determine fracture rate and time to fracture of dogs receiving RT (coarse or fine fractionated) for appendicular OS. Secondary objectives were to evaluate tolerability and disease outcome measures. METHODS: Fifty-one dogs that received RT as part of treatment for appendicular OS were available for evaluation. Forty-five received coarse fractionation (C-RT, 8 or 6 Gy per fraction protocols [C-RT8 or C-RT6]) while the remaining six received fine fractionation (F-RT). RESULTS: The overall pathologic fracture rate was 37%. Pathologic fracture rate was significantly higher for dogs that received F-RT (5/6, 83%) compared to dogs that received C-RT (12/40, 30%, p = 0.021). In the 17 dogs that fractured, the overall median time to fracture was 57 days. For all dogs, the median progression free interval (PFI) and median overall survival time (OST) were 90 and 140 days, respectively. In a very small cohort of dogs (n = 7) treated with zoledronate and RT, fracture rate was 0% and extended survival times were noted. CONCLUSIONS: In conclusion, C-RT is recommended over F-RT due to lower risk of pathologic fracture and similar PFI. Prospective evaluation of combined C-RT and zoledronate, especially for dogs with poor surgical candidacy, is warranted for the treatment of canine appendicular osteosarcoma.

Paired 18F-Fluorodeoxyglucose (18F-FDG), and 64Cu-Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (64Cu-ATSM) PET Scans in Dogs with Spontaneous Tumors and Evaluation for Hypoxia-Directed Therapy.

Hypoxia is associated with neoplastic tissue, protecting cancer cells from death by irradiation and chemotherapy. Identification of hypoxic volume of tumors could optimize patient selection for hypoxia-directed medical, immunological, and radiation therapies. Clostridium novyi-NT (CNV-NT) is an oncolytic bacterium derived from attenuated wild-type Clostridium novyi spores, which germinates exclusively in the anaerobic core of tumors with low-oxygen content. The hypothesis was that 64Cu-ATSM would localize to regions of hypoxia, and that greater hypoxic volume would result in greater germination of Clostridium novyi-NT (CNV-NT). Tumor-bearing companion dogs were recruited to a veterinary clinical trial. Dogs received a CT scan, 18F-FDG PET scan (74 MBq) and 64Cu-ATSM PET scan (74 MBq). Scan regions of interest were defined as the highest 20% of counts/voxel for each PET scan, and regions with voxels overlapping between the two scans. Maximum standardized uptake value (MaxSUV) and threshold volume were calculated. Direct oximetry was performed in select tumors. Tumor types evaluated included nerve sheath tumor (10), apocrine carcinoma (1), melanoma (3) and oral sarcoma (6). MaxSUVATSM ranged from 0.3-6.6. Measured oxygen tension ranged from 0.05-89.9 mmHg. Inverse of MaxSUVATSM had a linear relationship with oxygen tension (R2 = 0.53, P = 0.0048). Hypoxia <8 mmHg was associated with an SUVATSM > 1.0. Hypoxic volume ranged from 0 to 100% of gross tumor volume (GTV) and MaxSUVATSM was positively correlated with hypoxic volume (R = 0.674; P = 0.0001), but not GTV (P = 0.182). Tumor hypoxic volume was heterogeneous in location and distribution. 64Cu-ATSM-avid regions were associated with differential CT attenuation. Hypoxic volume did not predict CNV-NT germination. 64Cu-ATSM PET scanning predicts hypoxia patterns within spontaneously occurring tumors of dogs as measured by direct oxymetry. Total tumor volume does not accurately predict degree or proportion of tumor hypoxia.

A Pilot Study of Cancer-Induced Bone Pain Using Validated Owner Questionnaires, Serum N-Telopeptide Concentration, Kinetic Analysis, and PET/CT.

Cancer-induced bone pain, despite its frequency and severity, is a poorly understood phenomenon in people and animals. Despite excitement regarding translational osteosarcoma studies, there is a lack of attention toward examining cancer pain in dogs. In this pilot study, we used a multimodal pain assessment methodology to evaluate pain relief after therapeutic intervention in dogs with primary bone cancer. We hypothesized that intervention would cause objective evidence of pain relief. Evaluations of 8 dogs with primary bone cancer included 18F-FDG PET/CT scans, kinetic analysis, validated owner questionnaires (Canine Brief Pain Inventory, canine BPI), and serum N-telopeptide (NTx) concentration. Dogs were routinely staged and had 18F-FDG PET/CT scans prior to treatment with day 0, 7, 14, and 28 canine BPI, serum NTx, orthopedic exam, and kinetic analysis. Dogs treated with zoledronate and radiation underwent day 28 18F-FDG PET scans. All clinical trial work was approved by the University of Missouri IACUC. Four dogs underwent amputation (AMP) for their appendicular bone tumors; four received neoadjuvant zoledronate and hypofractionated radiation therapy (ZOL+RT). Canine BPI revealed significant improvements in pain severity and pain interference scores compared to baseline for all dogs. Positive changes in peak vertical force (+16.7%) and vertical impulse (+29.1%) were noted at day 28 in ZOL+RT dogs. Dogs receiving ZOL+RT had a significant (at least 30%) reduction in serum NTx from baseline compared to amputated dogs (p = 0.029). SUVmax (p = 0.11) and intensity (p = 0.013) values from PET scans decreased while tumor uniformity (p = 0.017) significantly increased in ZOL+RT-treated tumors; gross tumor volume did not change (p = 0.78). Owner questionnaires, kinetic analysis, and 18F-FDG PET/CT scans showed improved pain relief in dogs receiving ZOL+RT. Serum NTx levels likely do not directly measure pain, but rather the degree of systemic osteoclastic activity. Larger, prospective studies are warranted to identify the ideal objective indicator of pain relief; however, use of multiple assessors is presumably best. With improved assessment of pain severity and relief in dogs with cancer, we can better evaluate the efficacy of our interventions. This could directly benefit people with cancer pain, potentially decreasing the amount of subtherapeutic novel drugs entering human clinical trials.

A 3D-printed Apparatus for Imaging Multiple Rats Simultaneously.

CT (computerized tomography) is a necessary imaging modality for cancer staging and disease monitoring. Rodent models of cancer are commonly studied prior to human clinical trials, but CT in rodents can be difficult due to their small size and constant movement, which necessitates general anesthesia. Because microCT equipment is not always available, clinical CT may be a viable alternative. Limitations of microCT and clinical CT include biosecurity, anesthesia to limit image distortion due to motion, and cost. To address several of these constraints, we created a 3D-printed apparatus that accommodated simultaneous imaging of as many as 9 rats under gas anesthesia. Rats were anesthetized in series and placed in a 3 × 3 arrangement. To assess differences in attenuation between individual chambers and rows or columns in the device, we first imaged a standardized phantom plug as a control. We hypothesized that attenuation of specific rat organs would not be affected regardless of the location or position in the 3D-printed device. Four organs-liver, kidney, femur, and brain-were evaluated in 9 rats. For both the phantom and kidneys, statistically significant, but clinically negligible, effects on attenuation were noted between rows but not between columns. We attribute this finding to the absence of a top layer of the apparatus, which thus created asymmetric attenuation and beam hardening through the device. This apparatus allowed us to successfully image 9 rats simultaneously in a clinical CT machine, with negligible effects on attenuation. Planned improvements in this apparatus include completely enclosed versions for biosecure imaging.

Direct Interstitial Treatment of Solid Tumors Using an Injectable Yttrium-90-Polymer Composite.

Purpose: Yttrium-90 (90Y)-polymer composite (radiogel) may be administered directly into cancerous tissues to deliver highly localized beta radiation for therapy. In a dose-escalation study, the authors investigated the feasibility of treating feline and canine soft-tissue sarcomas as a model for nonresectable solid tumors in humans to gain clinical experience and to identify optimal methods for placing the composite uniformly within target tumor tissue. Materials and Methods: Five cats (Washington State University) and three dogs (University of Missouri) were selected for treatment from among veterinary clinic patients presenting with subcutaneous soft-tissue sarcomas. The therapeutic radiogel composite comprised two parts that were combined before therapy: (1) a calibrated activity of highly insoluble 90Y(YPO4) particles in a sterile, phosphate-buffered saline solution and (2) a resorbable hydrogel delivery vehicle containing a dissolved copolymer of poly-(DL-lactic acid-co-glycolic acid) and poly-(ethylene glycol). Sarcomas of anesthetized animals (five cats and three dogs) were injected with the 90Y-radiogel (10%-15% by tumor volume) using a parallel-needle grid pattern with ∼4-5-mm spacings with or without ultrasound guidance. After injection, the composite solution gelled within tumor interstitial spaces to solid phase upon reaching body temperatures to constrain the 90Y activity intratumorally. The animals were then imaged with computed tomography (CT) or positron emission tomography (PET)/CT and placed in radiation isolation for overnight monitoring and follow-up. Results: Gelation of the composite within tumor extracellular spaces confined the 90Y(YPO4) particles in place to deliver a planned radiation absorbed dose (100-320 Gy) to target tissue through complete decay. Response of the tumor tissue to 90Y-radiation therapy postexcision was evaluated by imaging, tumor resection, and histology. Correlation was observed on histopathology between tumor destruction and radiation dose. With uniform placement at high dose, the authors achieved complete remission or stable disease (at 1-2 months posttreatment). Conclusions: This study demonstrated successful injection of 90Y-polymer composite (radiogel) without discernable radiation dose to normal organs or other detrimental side effects. Animal patients recovered quickly from the injection procedure. The better therapeutic responses were observed at mean doses at or above 300 Gy.