Browsing by Author "Klahn, Shawna L."

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    ACVIM small animal consensus statement on safe use of cytotoxic chemotherapeutics in veterinary practice
    Smith, Annette N.; Klahn, Shawna L.; Phillips, Brenda; Parshley, Lisa; Bennett, Peter; Flory, Andi; Calderon, Rosemary (American College of Veterinary Internal Medicine, 2018-05)
    The purpose of this report is to offer a consensus opinion of ACVIM oncology diplomates and technicians on the safe use of cytotoxic chemotherapeutics in veterinary practice. The focus is on minimizing harm to the personnel exposed to the drugs: veterinary practitioners, veterinary technicians, veterinary staff, and pet owners. The safety of the patient receiving these drugs is also of paramount importance, but is not addressed in this statement. Much of the information presented is based on national recommendations by Occupational Safety and Health Administration, National Institute for Occupational Safety and Health, United States Pharmacopeia, and other published regulations. These directives reflect an abundance of caution to minimize exposure to medical personnel, but large-scale studies about the consequences of long-term occupational exposure are not available in veterinary medicine. Challenges in the delivery of optimal treatment safely and economically to veterinary patients in general practice without access to a veterinary oncologist or other specialist, because of costs or proximity, remain.
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    Characterization of Biomedical and Incidental Nanoparticles in the Lungs and Their Effects on Health
    McDaniel, Dylan K. (Virginia Tech, 2018-11-20)
    Nanomaterials are defined as any material with at least one external dimension less than 100 nm. Recently, nanomaterials have become more common in medicine, technology, and engineering. One reason for their increased interest is due to nanomaterials having unique properties that allow them to interact effectively with biological systems. In terms of drug delivery, the lungs are a highly desirable site to administer therapeutic nanoparticles. Indeed, inflammatory diseases such as asthma and emphysema could potentially benefit from nanoparticle-mediated delivery. However, the lungs are also in constant contact with airborne particulate matter. Thus, harmful nanoparticles can enter the lungs and cause or even exacerbate inflammatory diseases. Our work focused on characterization of both therapeutic and potentially harmful nanoparticles in the lungs. We found that fluorescently-labeled nanoparticles were phagocytosed by macrophages and did not induce apoptosis or inflammation in the lungs, making them potentially useful as a therapeutic for inflammatory diseases. We also characterized a rare form of titanium-based particles called Magnéli phases, which have been shown to be produced via coal burning. We found that while these particles are non-inflammatory in the lungs of mice, they lead to apoptosis of macrophages as well as a change in gene expression associated with increased fibrosis. Ultimately, this was shown to lead to a decrease in lung function parameters and airway hyperresponsiveness, indicating increased lung stiffness after long-term nanoparticle exposure. Our data adds significant contributions to the field by assessing two nanoparticles with vastly different compositions in the lungs. Overall, we found that the unique properties of both particle types allows for interactions with cells and tissues. These interactions can have important outcomes on health, both in terms of disease treatment and exacerbation.
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    Clinical outcomes in dogs with localized splenic histiocytic sarcoma treated with splenectomy with or without adjuvant chemotherapy
    Latifi, Max; Tuohy, Joanne L.; Coutermarsh-Ott, Sheryl; Klahn, Shawna L.; Leeper, Haley; Dervisis, Nikolaos G. (Wiley, 2020-09-28)
    Background: Localized splenic histiocytic sarcoma (HS) in dogs is a poorly understood disease, and could have longer survival times than disseminated or hemophagocytic HS. Understanding the clinical behavior of localized splenic HS can refine treatment recommendations. Objective: To describe the clinical characteristics and outcomes of dogs with localized splenic HS. Animals: Fourteen client-owned dogs with histologically confirmed splenic HS that received splenectomy. Methods: Multi-institutional retrospective case series—medical records of dogs with splenic HS were reviewed. Dog signalment, clinicopathologic data, primary and adjuvant treatments, and outcomes were obtained. Survival data were calculated using Kaplan-Meier analysis. Dog variables such as age, weight, platelet counts were reported using descriptive statistics. The Cox proportional hazards regression method was used to determine whether potential risk factors (weight, age, albumin level, hematocrit, and platelet count) were associated with PFI. Results: Median survival time for the dogs in this study was 427 days. Twelve dogs received adjuvant lomustine-based chemotherapy. Five dogs (35.7%) were suspected or confirmed to have developed metastatic disease. Eleven dogs died of disease, 1 dog died of unrelated cause, and 2 dogs were alive at final follow-up. Conclusions and Clinical Significance: Histiocytic sarcoma in dogs can manifest as a localized form in the spleen. Dogs with localized splenic HS treated with surgery ± chemotherapy can experience survival times over a year.
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    Detection of Cell-free Tumor DNA in Liquid Biopsies of Dogs with B cell Lymphoma: A Biomarker Discovery
    Vadlamudi, Sai Navya (Virginia Tech, 2024-08-12)
    Lymphoma is a common hematopoietic malignancy in canines. Current diagnostic techniques to diagnose lymphoma are often invasive and expensive. Additionally, tumor heterogeneity complicates the accurate classification and diagnosis of specific subtypes, hindering the development of targeted therapy and prognostic assessments. We propose a minimally invasive liquid biopsy technique involving blood collection to detect cell-free DNA from tumors using Next-generation sequencing. We hypothesize that identical tumor aberrations can be found in matching plasma and tumor DNA. Five dogs diagnosed with B-cell lymphoma through flow cytometry or PAAR were enrolled in the study. Samples collected included: (1) blood for plasma (cfDNA), (2) tumor tissue fine-needle aspirates (tumor DNA), and (3) buccal swabs (genomic DNA, germline control). Whole Genome Sequencing was performed using Illumina NovaSeq 6000, and the sequenced output was analyzed with bioinformatics tools to detect somatic variants in plasma and tumor samples. Our results revealed many shared somatic variants between matched cfDNA and tumor DNA samples, with 1.7-49% of tumor variants also found in corresponding plasma samples. Shared variants constituted only 0.5-9% of all plasma somatic variants. Specific B-cell lymphoma mutations were identified in cfDNA, including MYC, POT1, and TRAF3, alongside other cancer-related genes. Tumor samples showed mutations in genes associated with canine and human B-cell lymphoma. This study suggests that tumor-specific genomic mutations can be detected in plasma, supporting the potential of liquid biopsy as a less invasive diagnostic tool. However, cfDNA may not capture the full genetic heterogeneity of tumors due to low tumor-derived DNA content in limited plasma volumes.
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    Evaluation and comparison of outcomes in dogs with periarticular and nonperiarticular histiocytic sarcoma
    Klahn, Shawna L.; Kitchell, Barbara E.; Dervisis, Nikolaos G. (American Veterinary Medical Association, 2011-07-01)
    Objective—To evaluate and compare the outcomes of dogs with periarticular histiocytic sarcoma (PAHS) and histiocytic sarcoma of other anatomic locations (non-PAHS) and identify factors associated with outcome for dogs with PAHS. Design—Retrospective cohort study. Animals—19 dogs with PAHS and 31 dogs with non-PAHS. Procedures—Medical records of dogs with histiocytic sarcoma that underwent definitive local treatment (surgery or radiation), chemotherapy, or a combination of these were reviewed. Patient signalment, clinical signs, staging test results, clinicopathologic data, type of treatment, response, and outcome were collected, and potential risk factors in dogs with PAHS were identified and analyzed for an association with outcome. Results—Dogs with PAHS lived significantly longer than did dogs with non-PAHS, with an overall median survival times of 391 (range, 48 to 980) and 128 (range, 14 to 918) days, respectively, despite the presence of suspected metastasis at diagnosis in 13 of 19 dogs with PAHS. Dogs with PAHS without evidence of metastasis at diagnosis lived significantly longer than did dogs with PAHS with evidence of metastasis, with median survival times of 980 (range, 83 to 980) and 253 (range, 48 to 441) days, respectively. Administration of prednisone in dogs with PAHS was associated with a significantly shorter time to tumor progression (TTP) and increased risk of tumor progression and death. Conclusions and Clinical Relevance—Results indicated that dogs with PAHS may have a favorable outcome independent of metastatic status when treated with chemotherapy or aggressive multimodal treatment. The concurrent administration of prednisone may be a negative predictive factor for survival time and TTP in dogs with PAHS.
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    Evaluation of the therapeutic potential of Akt inhibition in a translational model of histiocytic sarcoma
    Qin, Qizhi (Virginia Tech, 2018-10-12)
    Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma. We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib. In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors.
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    Evaluation of Tumor Grade and Proliferation Indices before and after Short-Course Anti-Inflammatory Prednisone Therapy in Canine Cutaneous Mast Cell Tumors: A Pilot Study
    Klahn, Shawna L.; Dervisis, Nikolaos G.; Lahmers, Kevin K.; Benitez, Marian (MDPI, 2022-06-07)
    Glucocorticoid administration is a common clinical practice that attempts to decrease the inflammation associated with and improve the resectability of canine mast cell tumors (MCTs). However, the impact of neoadjuvant glucocorticoids on the histological features and proliferation indices of canine MCTs is unknown. The objective of this study was to evaluate changes in tumor grade, mitotic count, Ki67, AgNOR, and AgNORxKi67 scores following short-course anti-inflammatory neoadjuvant prednisone in canine patients with MCTs. This was a prospective single-arm pilot study. Client-owned dogs with treatment-naïve cytologically confirmed MCTs were enrolled. Patients underwent an initial incisional biopsy followed by a 10–14-day course of anti-inflammatory prednisone and surgical resection. All histological samples were randomized, masked, and evaluated by a single pathologist. Unstained paired pre- and post-treatment samples were submitted to a commercial laboratory for Ki67 and AgNOR immunohistochemical analysis. There were 11 dogs enrolled with 11 tumors. There were no statistical differences between the pre- and post-treatment histological parameters of mitotic index, Ki67, AgNOR, or Ki67xAgNOR. There were no clinically significant alterations between pre-treatment and post-treatment in the assignment of tumor grades. A short course of anti-inflammatory prednisone does not appear to alter the histological parameters that affect grade determination or significantly alter the proliferation indices in canine MCTs.
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    Hemophagocytic syndrome in a cat
    Wilkinson, Ashley R.; Carr, Susan V.; Klahn, Shawna L.; Dervisis, Nikolaos G.; Hanks, Cory R. (SAGE, 2018-07)
    Case summary: A 12-year-old male castrated domestic shorthair cat was evaluated for a 10 month history of weight loss. Thin body condition and a grade II/VI systolic parasternal heart murmur was noted during examination. Moderate-to-severe anemia and intermittent thrombocytopenia were identified on serial complete blood counts. Antibodies against feline immunodeficiency virus (FIV) were detected, but vaccination for FIV occurred previously. Echocardiography revealed biatrial and biventricular enlargement, left ventricular hypertrophy and pericardial effusion. Splenomegaly was present on abdominal ultrasound and cytological evaluation revealed macrophagic infiltration with erythrophagocytosis. Cytological evaluation of the bone marrow revealed similar findings. Histopathology of the spleen confirmed hemophagocytosis with no evidence of malignancy. A presumptive diagnosis of hemophagocytic syndrome was made. PCR testing for FIV on the splenic tissue was negative. The cat was treated with lomustine. Disease progression occurred approximately 6 months after diagnosis and the cat was euthanized. Relevance and novel information: To our knowledge, this is one of the few reports describing the diagnosis of hemophagocytic syndrome in a cat.
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    High Frequency Irreversible Electroporation (H-FIRE) as a Therapeutic Modality for Liver Cancer Treatment and Its Effect on the systemic Extracellular Vesicle Population
    Tellez Silva, Alejandra (Virginia Tech, 2024-08-02)
    High-frequency irreversible electroporation (H-FIRE) is a non-thermal ablation technique that uses intense, short, bipolar electrical pulses to induce cell death in cancerous tissues. It's being studied for treating hepatocellular carcinoma (HCC) in dogs. Previous in vitro research suggests H-FIRE may impact the release of extracellular vesicles (EVs). This study aims to explore how H-FIRE affects peripheral extracellular vesicle (EV) dynamics, potentially providing insights into its broader systemic effects and implications for biomarker development in canine liver cancer treatment. Dogs diagnosed with HCC were enrolled in a clinical trial. H-FIRE was applied to tumors, followed by surgical resection at three different time points. Peripheral blood samples were collected before and immediately after H-FIRE treatment. Plasma was isolated, aliquoted, and stored at -20°C. EVs were enriched from plasma via filtration and ultracentrifugation. Nanoparticle Tracking Analysis (NTA) quantified EV concentration and size distribution. Ten patients provided pre- and post-treatment plasma samples. The median EV concentration in peripheral blood increased from 2.56 x 10^11 particles/ml pre-treatment to 2.68 x 10^11 particles/ml post-treatment (p = 0.0048). The mean EV size decreased from 99.32 nm pre-treatment to 87.82 nm post-treatment (p = 0.007). The mode of EV size decreased from 83 nm pre-treatment to 70.5 nm post-treatment (p = 0.0076). The results of this study raise intriguing questions on the significance of changes in extracellular vesicle size and concentration post-treatment, as well as the potential clinical implications of these changes.
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    High intensity focused ultrasound for the treatment of solid tumors: a pilot study in canine cancer patients
    Carroll, Jennifer; Coutermarsh-Ott, Sheryl; Klahn, Shawna L.; Tuohy, Joanne L.; Barry, Sabrina L.; Allen, Irving C.; Hay, Alayna N.; Ruth, Jeffrey; Dervisis, Nikolaos G. (Taylor & Francis, 2022-01)
    Purpose: To investigate the safety, feasibility, and outcomes of High-Intensity Focused Ultrasound (HIFU) for the treatment of solid tumors in a spontaneous canine cancer model. Methods: Dogs diagnosed with subcutaneous solid tumors were recruited, staged and pretreatment biopsies were obtained. A single HIFU treatment was delivered to result in partial tumor ablation using a commercially available HIFU unit. Tumors were resected 3-6 days post HIFU and samples obtained for histopathology and immunohistochemistry. Total RNA was isolated from paired pre and post treated FFPE tumor samples, and quantitative gene expression analysis was performed using the nCounter Canine IO Panel. Results: A total of 20 dogs diagnosed with solid tumors were recruited and treated in the study. Tumors treated included Soft Tissue Sarcoma (n = 15), Mast Cell Tumor (n = 3), Osteosarcoma (n = 1), and Thyroid Carcinoma (n = 1). HIFU was well tolerated with only 1 dog experiencing a clinically significant adverse event. Pathology confirmed the presence of complete tissue ablation at the HIFU targeted site and immunohistochemistry indicated immune cell infiltration at the treated/untreated tumor border. Quantitative gene expression analysis indicated that 28 genes associated with T-cell activation were differentially expressed post-HIFU. Conclusions: HIFU appears to be safe and feasible for the treatment of subcutaneous canine solid tumors, resulting in ablation of the targeted tissue. HIFU induced immunostimulatory changes, highlighting the canine cancer patient as an attractive model for studying the effects of focal ablation therapies on the tumor microenvironment.
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    Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review
    Hendricks-Wenger, Alissa; Arnold, Lauren; Gannon, Jessica; Simon, Alex; Singh, Neha; Sheppard, Hannah; Nagai-Singer, Margaret A.; Imran, Khan Mohammed; Lee, Kiho; Clark-Deener, Sherrie; Byron, Christopher R.; Edwards, Michael R.; Larson, Martha M.; Rossmeisl, John H. Jr.; Coutermarsh-Ott, Sheryl; Eden, Kristin; Dervisis, Nikolaos G.; Klahn, Shawna L.; Tuohy, Joanne L.; Allen, Irving C.; Vlaisavljevich, Eli (IEEE, 2021-09-03)
    New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.
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    Histotripsy Ablation of Bone Tumors: Feasibility Study in Excised Canine Osteosarcoma Tumors
    Arnold, Lauren; Hendricks-Wenger, Alissa; Coutermarsh-Ott, Sheryl; Gannon, Jessica; Hay, Alayna N.; Dervisis, Nikolaos G.; Klahn, Shawna L.; Allen, Irving C.; Tuohy, Joanne L.; Vlaisavljevich, Eli (Elsevier, 2021-12)
    Osteosarcoma (OS) is a primary bone tumor affecting both dogs and humans. Histotripsy is a non-thermal, non-invasive focused ultrasound method using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the feasibility of treating primary OS tumors with histotripsy using a 500-kHz transducer on excised canine OS samples harvested after surgery at the Veterinary Teaching Hospital at Virginia Tech. Samples were embedded in gelatin tissue phantoms and treated with the 500-kHz histotripsy system using one- or two-cycle pulses at a pulse repetition frequency of 250 Hz and a dosage of 4000 pulses/point. Separate experiments also assessed histotripsy effects on normal canine bone and nerve using the same pulsing parameters. After treatment, histopathological evaluation of the samples was completed. To determine the feasibility of treating OS through intact skin/soft tissue, additional histotripsy experiments assessed OS with overlying tissues. Generation of bubble clouds was achieved at the focus in all tumor samples at peak negative pressures of 26.2 ± 4.5 MPa. Histopathology revealed effective cell ablation in treated areas for OS tumors, with no evidence of cell death or tissue damage in normal tissues. Treatment through tissue/skin resulted in generation of well-confined bubble clouds and ablation zones inside OS tumors. Results illustrate the feasibility of treating OS tumors with histotripsy. CORRIGENDUM: The authors regret that errors were present in the above article. The legend for Figure 5 on page 3441 should read “Fig. 5. Normal, healthy, non-neoplastic bone was excised from amputated canine limbs and subjected to histology. No histological differences were noted between untreated (a: magnification 4 x, b: magnification 40 x) and treated samples (c: magnification 4 x, d: magnification 40 x).” Also, the final section heading on page 3439 should read “Histotripsy ablation of ex vivo bone and nerve specimens.” Finally, the reference after the last complete sentence on page 3437 is incomplete and should read “Focal pressure waveforms for the 500-kHz transducer were measured using a custom-built fiberoptic hydrophone (FOPH) in degassed water at the focal point of each transducer (Parsons et al. 2006).” The authors would like to apologise for any inconvenience caused.
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    Identification of the presence and activity of the JAK-STAT pathway in canine solid tumors
    Fagan, Erin A. (Virginia Tech, 2017-05-22)
    Background: The JAK-STAT pathway is a cellular signaling pathway, which acts normally in humans and animals in the control of multiple important functions. Dysregulation of this pathway has been identified in human cancers, as well as a limited number of veterinary cancers. Objectives: The aims of this study were to identify the presence and tentative activity of components of the JAK-STAT pathway in selected canine tumors. Methods: Formalin-fixed, paraffin-embedded samples from mast cell tumors (MCT), hemangiosarcomas (HSA), thyroid carcinomas, and apocrine gland anal sac adenocarcinomas (AGASACA) were obtained from the Diagnostic Histopathology Laboratory at the Virginia Maryland College of Veterinary Medicine. Immunohistochemistry was performed to evaluate protein levels of JAK1, phospho-JAK1, JAK2, phospho-JAK2, STAT3, and phospho-STAT3. Signalment, treatment information, and survival information was obtained from the medical record for each case. Results: Tumor samples were scored for percent positive neoplastic cells. Positive staining was seen for all antibodies in all tumor types, with expression of JAK1, STAT3, and pSTAT3 being highest overall for all tumor types. Significant associations were seen between JAK1 and survival time in MCT (p = 0.03), pJAK1 and survival time in HSA (p = 0.009) and MCT (p = 0.04), and pSTAT3 and metastasis in MCT (p = 0.0008). Conclusions: The finding of positive staining for the components of the JAK-STAT pathway in the tumor samples evaluated indicates presence and tentative activity of this pathway in the studied cancers. Further study of JAK1, pJAK1, and pSTAT3 should be pursued to evaluate their potential as therapeutic targets.
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    Immune Checkpoint Molecule Expression in Canine Lymphoma and Canine Reactive Lymphoid Hyperplasia
    Clothier, Stacy Lauren (Virginia Tech, 2019-11-12)
    Background: Although lymphoma is one of the most common malignancies in dogs, remission rates and survival times remain stagnant. Treatment with a multi-agent chemotherapy protocol induces remission for less than one year and the majority of patients relapse. Fewer than 25% of dogs live longer than two years with the currently available treatments. Targeted immunotherapy using checkpoint molecule blockade of PD-1 and PD-L1 shows promise for various types of human cancer, including relapsed/refractory lymphoma; however, little is known regarding the role of these checkpoint molecules in canine lymphoma. Objectives: To determine the patterns of expression of mRNAs encoding PD-1 and its ligands PD-L1 and PD-L2 in lymphoma and reactive lymphoid hyperplasia controls. Methods: Retrospective: formalin-fixed paraffin-embedded (FFPE) tissue from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Prospective: fine-needle aspirates (FNAs) from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Total RNA was extracted, and expression of PD-1, PD-L1, and PD-L2 was measured using qRT-PCR analysis of random-primed cDNA. Checkpoint molecule expression levels were determined using the 2^∆∆CT method. Lymphoma immunophenotype was assessed using immunohistochemical analysis of CD3 and CD79a (FFPE) and review of patient medical records (FNA). Data analysis included Wilcoxon ranksum tests, Dunn's procedure of multiple comparisons, Kruskal-Wallis tests, and regression within an ANOVA. Significance at P < 0.05. Results: PD-1, PD-L1, and PD-L2 expression (normalized internally to 18S rRNA) was lower in lymphoma compared to reactive lymphoid hyperplasia (FFPE); the difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. PD-1, PD-L1, and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FFPE); this difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. The higher relative abundance of PD-L1 vs PD-1 and PD-L2 vs PD-1 was significantly different between lymphoma and reactive lymphoid hyperplasia (FFPE and FNA). Conclusions: In this study, checkpoint molecule expression was not upregulated in canine lymphoma relative to canine reactive lymphoid hyperplasia, suggesting a limited application of PD-1 and PD-L1 blockade in canine lymphoma. The ligand:receptor relative abundance imbalances reflect the lower PD-1 expression relative to PD-L1 and PD-L2 in lymphoma. Although these results do not suggest that checkpoint inhibitors would be useful for treatment, they give insight into the mechanisms of unchecked lymphocyte proliferation in canine lymphoma.
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    Investigations of Ultrasound-Guided Histotripsy Ablation for Soft Tissue Sarcomas, Osteosarcomas, and Brain Tumors
    Ruger, Lauren N. (Virginia Tech, 2023-05-16)
    Histotripsy is a non-thermal, non-invasive focused ultrasound therapy using controlled acoustic cavitation to mechanically disintegrate tissue into an acellular homogenate. Histotripsy applies microsecond-length, high pressure (> 10 MPa) pulses to initiate the rapid expansion and collapse of nuclei in a millimeter-scale focal region, applying large stresses and strains to targeted tissues. The cavitation "bubble cloud" generated during histotripsy treatment can be visualized in real time on ultrasound imaging, assisting with treatment guidance and monitoring. Past studies have demonstrated histotripsy's potential for a variety of applications, but histotripsy has not yet been investigated for superficial musculoskeletal tumor ablation. Additionally, preliminary investigations using histotripsy to ablate brain tumors are underway, but require advanced histotripsy devices capable of overcoming attenuation of the therapeutic ultrasound signal by the skull and rely on MRI for real-time guidance. As a result, open questions remain regarding ultrasound-guided histotripsy for brain tumors. Early evidence also suggests that histotripsy ablation may induce immunogenic changes in the tumor microenvironment. Continued research is needed to explain and corroborate these findings under conditions more immunologically representative of human cancers, such as in large animal models with spontaneous tumors. This dissertation investigates the safety and feasibility of using ultrasound-guided histotripsy to ablate superficial soft tissue sarcomas (STS), osteosarcomas (OS), and brain tumors and considers the immunological impacts of histotripsy treatment for STS and OS. The research described herein (1) investigates the ability of histotripsy to treat superficial STS tumors in companion animals with spontaneous tumors, (2) investigates the feasibility of treating bone tumors with histotripsy through a series of ex vivo and in vivo studies, and (3) applies histotripsy for the minimally invasive treatment of superficial brain tumors. The completion of this dissertation will provide significant insight into the ability of ultrasound-guided histotripsy to treat novel tumor types (i.e., STS, OS, and brain tumors) and the potential role of histotripsy in veterinary medicine. Future work will build upon the studies detailed in this dissertation to optimize ultrasound-guided histotripsy for the treatment of complete STS, OS, and brain tumors in veterinary and human patients.
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    Invited Review-Neuroimaging Response Assessment Criteria for Brain Tumors in Veterinary Patients
    Rossmeisl, John H. Jr.; Garcia, Paulo A.; Daniel, Gregory B.; Bourland, John Daniel; Debinski, Waldemar; Dervisis, Nikolaos G.; Klahn, Shawna L. (Wiley-Blackwell, 2014-03-01)
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    Mechanical High-Intensity Focused Ultrasound (Histotripsy) in Dogs with Spontaneously Occurring Soft Tissue Sarcomas
    Ruger, Lauren N.; Yang, Ester; Gannon, Jessica; Sheppard, Hannah; Coutermarsh-Ott, Sheryl; Ziemlewicz, Timothy J.; Dervisis, Nikolaos G.; Allen, Irving C.; Daniel, Gregory B.; Tuohy, Joanne L.; Vlaisavljevich, Eli; Klahn, Shawna L. (IEEE, 2023-03)
    Introduction: Histotripsy is a non-invasive focused ultrasound therapy that uses controlled acoustic cavitation to mechanically disintegrate tissue. To date, there are no reports investigating histotripsy for the treatment of soft tissue sarcoma (STS). Objective: This study aimed to investigate the in vivo feasibility of ablating STS with histotripsy and to characterize the impact of partial histotripsy ablation on the acute immunologic response in canine patients with spontaneous STS. Methods: A custom 500 kHz histotripsy system was used to treat ten dogs with naturally occurring STS. Four to six days after histotripsy, tumors were surgically resected. Safety was determined by monitoring vital signs during treatment and post-treatment physical examinations, routine lab work, and owners’ reports. Ablation was characterized using radiologic and histopathologic analyses. Systemic immunological impact was evaluated by measuring changes in cytokine concentrations, and tumor microenvironment changes were evaluated by characterizing changes in infiltration with tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) using multiplex immunohistochemistry and differential gene expression. Results: Results showed histotripsy ablation was achievable and well-tolerated in all ten dogs. Immunological results showed histotripsy induced pro-inflammatory changes in the tumor microenvironment. Conclusion & Significance: Overall, this study demonstrates histotripsy's potential as a precise, non-invasive treatment for STS.
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    Mechanical High-Intensity Focused Ultrasound (Histotripsy) in Dogs with Spontaneously Occurring Soft Tissue Sarcomas
    Yang, Ester (Virginia Tech, 2023-09-06)
    Background: Histotripsy is a non-thermal, non-invasive high-intensity focused ultrasound (HIFU) ablative technique that causes mechanical fragmentation of tissue, resulting in liquefied acellular debris with histologically clear demarcated boundaries between treated and non-treated tissues. The acellular debris may include tumor antigens with preserved immunogenicity and the potential to generate systemic immune response against tumor cells. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to STS in humans. Long-term tumor control requires complete removal with extensive surgical resection, which in many cases is not feasible. As a result, there is need for alternative therapies. Objectives: The primary objective of this study was to demonstrate safety and feasibility of histotripsy in a small animal model of spontaneous STS. The secondary objective was to characterize the impact of histotripsy on the immunologic response. Materials and methods: Pet dogs diagnosed with spontaneous STS were recruited. CT scan of the chest, abdomen, and the tumor was performed for staging and treatment planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, owner reports, and CBC/serum biochemistry. Partial tumor ablation was performed using a 500 kHz prototype histotripsy system. A spherical treatment zone of up to 3 cm diameter in each tumor was treated with histotripsy according to the patient-specific treatment plan using 1-2 cycle pulses applied at a pulse repetition frequency (PRF) of 500 Hz. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-6 days post-treatment. Tumor microenvironment (TME) gene expression was evaluated with the Nanostring Canine IO panel, and the systemic immune response was evaluated using multiplex serum cytokine levels. Results: Ten dogs were recruited and treated. Tumor histologies included 3 grade III STS, 4 grade II STS, 2 grade I STS, and 1 malignant mesenchymoma. Six dogs were alive, three dogs were euthanized due to disease progression, and one dog was lost to follow up. Histotripsy-related complications were generally self-limiting, with only one patient having increased cutaneous injury score from 1 to 2 (scale 1-5) post-treatment, likely due to prefocal cavitation at the skin. No significant adverse events impacting patient outcome were noted in any of the patients. Visible histotripsy cavitation bubble clouds were seen on real-time ultrasound imaging in nine of ten treatments. Post-treatment histopathology indicated sharply defined regions of ablation that were clearly identifiable grossly and histologically in all samples. Treatment zones were characterized by loss of cell viability, hyalinization, and acute hemorrhage. Post-treatment contrast-enhanced CT images revealed clear, demarcated regions of histotripsy ablated tissue in seven of ten patients. Differential gene expression analysis identified 79 genes with at least 2-fold change following treatment. Genes associated with inflammation, immune cell migration, and immune cell interactions were the highest upregulated. Amongst the gene set analyses, the myeloid compartment gene sets obtained the highest significance score. There were no statistically significant differences between pre- and post-treatment cytokine concentrations for any of the analytes. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs diagnosed with STS. Histotripsy induced pro-inflammatory changes within the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Histotripsy has a potential to be a precise, non-invasive treatment for canine STS.
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    Molecular basis of immunotolerance in canine neoplasia
    Stevenson Salinas, Valentina Beatriz (Virginia Tech, 2023-01-30)
    Melanoma is a highly malignant neoplasia with high rates of metastasis in humans and dogs. Regardless of being considered a highly immunogenic neoplasm, the function of the immune system is hampered by the expression of immune checkpoint molecules by the cancer cells. In contrast, soft tissue sarcomas are poorly immunogenic, as Tumor infiltrating Lymphocytes are lacking, or when present they are usually at the periphery of the tumor. Still, soft tissue sarcomas are considered immunosuppressed. Checkpoint molecules from the PD-axis are overexpressed in numerous human malignant neoplasia and have recently gained attention with a few reports in canine tumors. Immunotherapies against these checkpoint molecules have shown great efficacy in humans, but in order to determine translational approaches into canine patients, more research is needed. Here we determined the gene expression of Programed Death receptor-1, and its ligands PD-L1 and PD-L2 in canine tumors with two distinct immune profiles. Our results show that regardless of their immune profiles, melanoma versus soft tissue sarcoma, checkpoint molecules expression was higher in malignant tumors with a higher grade. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal ultrasound focused therapy that induces mechanical stress to the cells, leading to liquefactive necrosis. Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of CD3+ T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasia, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia.
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    Overcoming therapeutic resistance in glioblastoma using novel electroporation-based therapies
    Partridge, Brittanie R. (Virginia Tech, 2022-10-25)
    Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy.