You’re There For Them and We’re Here For You

With so many unknowns in the world right now, you can rest comfortably knowing that Amatheon is here for you, as you have been here for us throughout the years. We are proud to have served the veterinary oncology community for the past 18 years. You can count on us to help you with all your chemotherapy, specialty and control medication needs, as well as oncology safety supplies.

CHEMOTHERAPY SPECIALTY MEDICATIONS CONTROL MEDICATIONS MEDICATIONS (C3-5 & C2)

Phaseal™ You’re There For Them and We’re Here For You Table of Contents

Welcome...... 2 Organizing & Executive Committees...... 3 Schedule at a Glance...... 4 Keynote Speakers...... 7 Resident Review Workshop...... 8 Schedule of Oral Scientific Presentations...... 9 Poster Abstract Listings...... 14 Specialty Sessions...... 31 Abstracts: With so many unknowns in the world right now, you can rest comfortably knowing that • Resident Oral Abstracts...... 55 Amatheon is here for you, as you have been here for us throughout the years. We are proud to • International Track Abstracts...... 65 have served the veterinary oncology community for the past 18 years. You can count on us to help you with all your chemotherapy, specialty and control medication needs, as well as • Oral Abstracts...... 81 oncology safety supplies. • Poster Abstracts...... 99 Veterinary Technician Program...... 139 Continuing Education Information...... 177 Author Index...... 178

Contact Information:

Veterinary Cancer Society PO Box 30855 SPECIALTY MEDICATIONS CONTROL MEDICATIONS CHEMOTHERAPY Columbia, MO 65205 MEDICATIONS (C3-5 & C2) Telephone 573-823-8497 Fax 573-445-0353 [email protected] vetcancersociety.org

Phaseal™ 1 Welcome to the 2020 VCS Annual Conference

Dear Friends and Colleagues,

What a year it has been! Unfortunately, we are unable to gather in Daytona Beach, Florida for our Annual Conference. Instead, we transitioned to a virtual Annual Conference through an online platform that will be available to you and all the rest of our oncology community from around the world. Nearly all of the presentations and sessions that you would have found in person will be brought to you virtually, both live online and on demand, at your leisure for up to a year. We hope this will become a way to feel connected to our community, learn at your own pace and discover technology and services from the industry.

Our keynote speakers are looking forward to meeting you virtually and include Elias Sayour, MD, PhD. Dr. Sayour is an Assistant Professor of Neurosurgery and Pediatrics and Principal Investigator of the RNA Engineering Laboratory within the Preston A. Wells, Jr. Center for Brain Tumor Therapy and University of Florida Brain Tumor Immunotherapy Program. Dr. Sayour trained at Duke University where he completed a pediatric hematology-oncology fellowship and NIH research fellowship in cancer biology and developmental therapeutics. He completed his PhD in 2015 at Duke University. Dr. Sayour will discuss his laboratory’s work and new approaches to immunotherapy of brain tumors and osteosarcoma, with a translational outlook on his research.

Mitchell S. Steiner, MD, FACS is a board certified urologist and a Fellow of the American College of Surgeons and has held numerous academic appointments, including Assistant Professor of Urology, Cell Biology, and Pathology at Vanderbilt School of Medicine from 1993 to 1995 and Chairman and Professor of Urology, Director of Urologic Oncology and Research and the Chair of Excellence in Urologic Oncology at the University of Tennessee from 1995 to 2004. Dr. Steiner holds a BA in Molecular Biology and Chemistry from Vanderbilt University and an MD from the University of Tennessee. Dr. Steiner is the President and Chief Executive Officer of VERU Inc., an oncology biopharmaceutical company developing and commercializing novel drugs. He will discuss the development of a novel oral selective antitubulin agent that targets alpha and beta tubulin of microtubules for advanced prostate cancer as well as for other forms of metastatic cancers, in addition to its application in comparative oncology.

You will also find a Resident Review Workshop on Thursday evening, concurrent specialty sessions on Friday and Saturday, a three-day technician workshop, abstract presentations and more. We look forward to your participation.

We are glad you can join us from the comfort of your own home or office and hope that you all stay healthy and safe until we can meet again in person.

Amandine Lejeune, DVM, DACVIM (Oncology)

2 Committees

Organizing Committee VCS Executive Committee Conference Chair President Amandine Lejeune, DVM Timothy Fan, DVM, PhD Diplomate ACVIM (Oncology) Diplomate ACVIM (Oncology/SAIM) University of Florida University of Illinois VTCS Workshop Coordinators President-Elect Aspen Schreiner, CVT Heather Wilson-Robles, DVM Southeast Veterinary Oncology & Internal Medicine Diplomate ACVIM (Oncology) Texas A&M University Casey Mobley, CVT Southeast Veterinary Oncology & Internal Medicine Treasurer Ira Gordon, DVM Resident Review Workshop Coordinator Diplomate ACVR (Radiation Oncology) Heather Wilson-Robles, DVM The Oncology Service Diplomate ACVIM (Oncology) Texas A&M University Secretary Valerie MacDonald-Dickinson, DVM Resident-Mentor Roundtable Coordinator Diplomate ACVIM (Oncology) Caroline Wood, DVM, PhD University of Saskatchewan University of Minnesota Members-at-Large Friday Concurrent Session Chairs Maria Dagli, DVM, MS, PhD Dr. Shea Cox, DVM, VCPP, CHPV University of Sao Paulo Dr. William Eward, DVM, MD Dr. Nicola Mason, B.VetMed, PhD, DACVIM (IM) Amandine Lejeune, DVM Dr. Siobhan Haney, VMD, MS, DACVR (RO) Diplomate ACVIM (Oncology) Dr. Cheryl London, DVM, PhD, DACVIM (Onc) University of Florida Dr. Robert Rebhun, DVM, PhD, DACVIM (Onc) Sandra Bechtel, DVM Audio Visual and IT Services Diplomate ACVIM (Oncology) Lee Fent University of Florida Live It Now Entertainment Katie Curran, DVM, MS Veterinary Cancer Society Diplomate ACVIM (Oncology) Sandi Strother, Executive Director Oregon State University Cindy Hazelrigg, Associate Director Resident Member-at-Large Sharon Rodes, Assistant Caroline Wood, DVM, PhD University of Minnesota Past-President Kim Selting, DVM Diplomate ACVIM (Oncology) Diplomate ACVR (Radiation Oncology) University of Illinois

3 Schedule at a Glance

(Technician Workshop schedule located on page 139) Thursday, October 15, 2020 5:00 pm Virtual Exhibit Hall and Poster Hall Open 5:30 pm - 5:45 pm Conference Welcome and Virtual Conference Details 5:45 pm - 6:00 pm Sponsor Presentation by Wedgewood Pharmacy 6:00 pm - 8:40 pm Resident Review Workshops Sponsored by Wedgewood Pharmacy • 6:00 pm - 7:15 pm The concepts of cancer epigentics and their potential implications for veterinary patients Dr. Jeffrey Bryan • 7:25 pm - 8:40 pm Diagnosis and classification of canine and feline lymphoproliferative disorders Dr. Anne Avery

Friday, October 16, 2020 10:00 am - 10:30 am Virtual Exhibit Hall and Poster Viewing 10:30 am - 11:00 am Sponsor Presentation by Elanco Animal Health Tanovea and canine lymphoma Dr. Doug Thamm 11:00 am - 12:00 pm Resident and International Abstract Tracks 12:00 pm - 12:45 pm Virtual Exhibit Hall and Poster Viewing 12:45 pm - 2:00 pm Resident and International Abstract Tracks

2:00 pm - 2:30 pm Virtual Exhibit Hall and Poster Viewing 2:30 pm - 3:30 pm Keynote Presentation Sponsored by Pathway Vet Alliance VERU-111, an oral selective alpha and beta tubulin inhibitor, for the treatment of canine cancers Dr. Mitchell Steiner

3:30 pm - 4:00 pm Virtual Exhibit Hall and Poster Viewing

This conference takes place in Eastern Time.

4 Schedule at a Glance

Friday, October 16, 2020 continued 4:00 pm - 4:15 pm Sponsor Presentation by ImpriMed, Inc. ImpriMed: A data-driven, personalized chemotherapy drug testing service for canine blood cancer patients 4:15 pm - 7:15 pm Specialty Sessions • Mast Cell Tumors Session and Panel Discussion Sponsored by Antech Diagnostics • Radiation Oncology Session and Panel Discussion Sponsored by Varian • Immunotherapy Session and Panel Discussion Sponsored by Elias Animal Health

Saturday, October 17, 2020 10:00 am - 10:30 am Virtual Exhibit Hall and Poster viewing 10:30 am - 11:00 am Sponsor Presentation by Virbac Tigilanol tiglate: Timely tool for the tumor toolbox 11:00 am - 12:00 pm General Abstract Tracks 12:00 pm - 12:45 pm Virtual Exhibit Hall and Poster Viewing 12:45 pm - 1:00 pm Sponsor Presentation by Verasonics, Inc. 1:00 pm - 2:00 pm Keynote Presentation Sponsored by Pathway Vet Alliance Cancer immunotherapy with nanoparticles: From canines to humans Dr. Elias Sayour 2:00 pm - 3:30 pm General Abstract Tracks 3:30 pm - 4:00 pm Virtual Exhibit Hall and Poster Viewing 4:00 pm - 4:15 pm Sponsor Presentation by Companion Animal Health Use of gold nanoparticles & laser to treat cancer: An update

This conference takes place in Eastern Time.

5 Schedule at a Glance

Saturday, October 17, 2020 continued 4:15 pm - 7:25 pm Specialty Sessions • Lymphoma Session and Panel Discussion Sponsored by Elanco Animal Health • Surgical Oncology Session and Panel Discussion Sponsored by Elias Animal Health • Palliative Care/Alternative Therapies 7:30 pm - 8:30 pm Closing Event, Awards and Membership Meeting

This conference takes place in Eastern Time.

6 Keynote Speakers

Sponsored by

Friday, October 16 | 2:30 pm Mitchell Steiner, MD VERU-111, an oral selective alpha and beta tubulin inhibitor, for the treatment of canine cancers Antitubulins, like taxanes, are the most widely used and effective agents in human oncology. Unfortunately, antitubulins have major limitations in canine oncology care: intravenous route of administration, high rates of severe hypersensitivity reactions to the cremophor EL solvent, and dose limiting toxicities of severe febrile neutropenia and sepsis. VERU-111 is an oral antitubulin that targets both alpha and beta tubulin subunits of microtubules and could have an important role against canine malignancies. Based on safety studies in dogs, VERU-111 has high oral bioavailability without the hypersensitivity, neurotoxicity and neutropenia dose limiting toxicities typical of taxanes. Preclinical in vitro cell line studies show that VERU-111 has activity against many of the common canine tumors including mast cell tumor, hemangiosarcoma, osteosarcoma, and lymphoid malignancies. A Phase 1 study is currently being planned in dogs with a variety of metastatic cancers. Oral antitubulin VERU-111 could be an important new addition to the armamentarium against canine cancers: ability for home administration and dose changes with efficacy against a broad ranges of cancer types and a better safety profile.

Saturday, October 17 | 1:00 pm Elias Sayour, MD, PhD Cancer immunotherapy with nanoparticles: From canines to humans Dr. Sayour’s translational efforts are also focused on new pipeline technologies including a novel lipid-nanoparticle (NP) formulation that his team has pioneered for the immunologic treatment of cancer. In this presentation, Dr. Sayour will discuss challenges to creating effective cancer vaccines, advantages of a nanoparticle based approach, and how the comparative oncology model has served as an important bridge to translate novel vaccine approaches. Dr. Sayour will discuss how the comparative oncology model can be leveraged for establishing safety, response correlates and activity of new therapies while simultaneously helping both pet dogs and humans with cancer.

This conference takes place in Eastern Time.

7 Resident Review Workshop

Thursday, October 15, 2020 l 6:00 pm – 8:40 pm Sponsored by

6:00 pm – 7:15 pm 7:25 pm – 8:40 pm Dr. Jeffrey Bryan Dr. Anne Avery University of Missouri Colorado State University The concepts of cancer Diagnosis and epigenetics and their classification of canine and potential implications feline lymphoproliferative for veterinary patients disorders Cancer is frequently called a genetic disease. What is Lymphoproliferative disorders are a highly heterogeneous often overlooked are the epigenetic contributions to group of diseases that arise from different stages of malignancies. Evidence exists that epigenetic changes lymphocyte development and have widely variable precede genetic mutations in many cancers. Most often clinical outcomes. The goal of this session is to provide in the cancers examined to date, epigenetic abnormalities, the audience with an understanding of the biological and or epimutations, greatly exceed sequence mutations in clinical behavior of a range of canine lymphoproliferative frequency. While mutations affect the genetic sequence, disorders. The emphasis of the session will be on the the hardware of the genetic code, epigenetic marks select most efficient and clinically useful method of diagnosis genes and chromosomal regions for transcriptional and classification. The advantages and limitations of activity, acting as the software that selects and interprets cytology, histology and immunohistochemistry, clonality the code to determine cell phenotype. Epigenetic testing and flow cytometry will be presented. While our changes may even lead directly to large mutational events understanding of feline lymphoproliferative disorders like translocations and contribute to selection of splice is less well-developed than in dogs, feline small cell variants of expressed genes. In this lecture, residents will intestinal lymphoma has been the focus of intense be taught the general forms of epigenetic changes that are research recently. Methods for diagnosing this disease known to contribute to gene expression, phenotype, and and controversies around those methods, will be also be the malignant condition. Fundamental concepts will be discussed. presented and will be supported by published research describing human cancers to illustrate classic principles. Epigenetic research in veterinary cancers will be presented to paint our current understanding of these principles in companion animal cancers. This conference takes place in Eastern Time.

8 Resident Oral Abstracts Friday, October 16, 2020

TIME PRESENTER ABSTRACT TITLES PAGE 10:00 am - 10:30 am EXHIBIT HALL/POSTER VIEWING 10:30 am - 11:00 am Dr. Doug Thamm Sponsor Presentation by Elanco Animal Health Tanovea-CA-1 Frequently Asked Questions 11:00 am - 11:15 am Samuel Keepman Safety evaluation of low dose meloxicam with toceranib 56 phosphate (Palladia) in cancer bearing cats: A Phase I dose-finding study 11:15 am - 11:30 am Gabrielle Carter Lomustine based chemotherapy protocols for the 57 treatment of canine T-cell lymphoma 11:30 am - 11:45 am Caroline Murray Outcome in 27 dogs with curative-intent treatment of 58 localized primary pulmonary histiocytic sarcoma 11:45 am - 12:00 pm Jennifer Carroll First in patient trial of high intensity focused ultrasound 59 (HIFU) for treatment of canine solid tumors 12:00 pm - 12:45 pm BREAK/EXHIBIT HALL/POSTER VIEWING 12:45 pm - 1:00 pm Kara Magee A multimodality radioimmunotherapy approach to 60 treating advanced stage cancer in companion dogs 1:00 pm - 1:15 pm Amanda Brehm Safety and feasibility of whole lung irradiation in the 61 treatment of canine appendicular osteosarcoma 1:15 pm - 1:30 pm Brittany Proteasome inhibition via bortezomib induces 62 Feldhaeusser Apoptosis in canine glioma cells 1:30 pm - 1:45 pm Christopher Characterizing circulating nucleosomes in the plasma of 63 Dolan dogs with lymphoma

1:45 pm - 2:00 pm Christopher PD-1 and PD-L1 expression on canine urothelial 64 Pinard carcinoma cell lines and the relative expression of lymphocyte PD-1 in canine urothelial carcinoma patients

See pages 5 for balance of the Friday schedule.

This conference takes place in Eastern Time.

9 International Abstracts Friday, October 16, 2020

The following abstracts were selected by an international committee representing ESVONC, JVCS, ABROVET and AMONCOVET, and were submitted for presentation at conferences which did not occur in 2020 due to the worldwide Covid-19 pandemic. VCS is proud to include the presentation of these outstanding oral abstracts during our Annual Conference.

TIME PRESENTERS ABSTRACT TITLES PAGE 11:00 am - 11:15 am Laurien Feenstra Minimal dose efficacy of PEG-L-Asparaginase in healthy 66 beagle dogs Eliza Canine transmissible venereal tumor: Retrospective study 67 Vazquez-Sanchez of 50 cases diagnosed during the period of 2016- 2019 in Mexico 11:15 am - 11:30 am Pierre Boyé Phase 1 dose escalation study of 12B80 – 68 hydroxybisphosphonate linked doxorubicin – in dogs with naturally occurring osteosarcoma Kazushi Asano En bloc resection of extensive adrenal 69 pheochromocytoma including the caudal vena cava in dogs 11:30 am - 11:45 am Taylor DePauw Machine learning for early detection of 70 hemangiosarcoma 11:45 am - 12:00 pm Sungwon Lim Predicting likelihood of in vivo chemotherapy response 72 in canine lymphoma using ex vivo drug sensitivity and immunophenotyping data in a machine learning model 12:00 pm - 12:45 pm BREAK/EXHIBIT HALL/POSTER VIEWING 12:45 pm - 1:00 pm Hirataka An exploratory investigation of novel molecular targets 74 Tomiyasu for chemotherapy in canine histiocytic sarcoma Luigi Aurisicchio Electro-immunotherapy in veterinary medicine: 75 Activating local and systemic immunity 1:00 pm - 1:15 pm Benoit Hédan Canine histiocytic sarcoma: From the genetic 76 predispositions and somatic alterations to the development of a genetic risk test and therapeutic options

This conference takes place in Eastern Time.

10 International Abstracts Friday, October 16, 2020

TIME PRESENTERS ABSTRACT TITLES PAGE 1:15 pm - 1:30 pm Anais Prouteau Circulating tumor DNA as a novel biomarker for 77 canine cancers: Histiocytic sarcoma, oral melanoma and lymphoma 1:30 pm - 1:45 pm Sabine Brandt Recombinant live-attenuated influenza viruses 78 co-expressing bovine papillomavirus 1 (BPV1) E6 and E7 induce tumour regression in equine sarcoid patients See pages 5 for balance of the Friday schedule.

This conference takes place in Eastern Time.

11 General Abstracts Saturday, October 17, 2020

TIME PRESENTERS ABSTRACT TITLES PAGE 10:30 am - 11:00 am Sponsor Presentation by Virbac Tigilanol tiglate: Timely tool for the tumor toolbox 11:00 am - 11:15 am Shunsuke Noguchi MIR-190A/NRG3/ERBB4 cascade as a potential 82 therapeutic target of canine glioma Gillian Dank Non-invasive detection of spontaneous tumors in 83 dogs 11:15 am - 11:30 am Anna Luiza Facchetti Stat3 pathway is upregulated in canine T-cell 84 Vinhaes Assumpcao lymphoma Heather Wilson-Robles Characterizing circulating nucleosomes in the 85 plasma of dogs with hemangiosarcoma 11:30 am - 11:45 am Margaret Musser Adverse event profile of the therapeutic canine 86 osteosarcoma vaccine, live listeria vector Jordan Ayers Novel application of single cell next-generation 87 sequencing for determination of intratumoral heterozygosity of canine osteosarcoma 11:45 am - 12:00 pm Lisa Parshley Nanoparticle and laser ablation of solid tumors in 88 dogs and cats Koangyong Sung Visualization and characterization of cancer stem 89 cells with low proteasome activity in a canine osteosarcoma cell line 12:45 pm - 1:00 pm Sponsor Presentation by Verasonics, Inc. 1:00 pm - 2:00 pm Keynote: Dr. Elias Sayour 7 2:00 pm - 2:15 pm Abby Reising Solitary osseous plasmacytomas in dogs: 90 A retrospective review of 13 cases Amandine Lejeune RNA in-situ hybridization as a molecular diagnostic 91 technique targeting IBA-1 and CD204 in canine histiocytic sarcoma 2:15 pm - 2:30 pm Constanza Pereira Retrospective study of feline mediastinal lymphoma: 92 Rivera Presentation and response to chemotherapy treatment Jordon Inkol In vitro effects of monoacylglycerol lipase inhibition 93 on phenotype in canine urothelial carcinoma cell lines

This conference takes place in Eastern Time.

12 General Abstracts Saturday, October 17, 2020

TIME PRESENTERS ABSTRACT TITLES PAGE 2:30 pm - 2:45 pm Hiroki Yamazaki Radiotherapy alone versus chemotherapy followed by 94 radiotherapy for cats with nasal lymphoma Gabriela Toledo Repurposing drugs in veterinary cancer treatment: 95 Sodium dichloroacetate and omeprazole exhibit synergistic cytotoxic effects on canine oral melanoma cell line 2:45 pm - 3:00 pm Alycen Lundberg Combining zoledronate with radiation therapy to 96 enhance the treatment of osteoinvasive feline oral squamous cell carcinoma 3:00 pm - 3:15 pm Hiroto Yoshikawa Retrospective evaluation of feline intranasal 97 carcinomas treated with external-beam radiotherapy: 43 cases 3:15 pm - 3:30 pm Kim Selting Kinetics of cardiac troponin 1 release and correlation 98 with echocardiographic and electrocardiographic parameters in dogs treated with single agent doxorubicin 4:00 pm - 4:15 pm Sponsor Presentation by Companion Animal Health Use of gold nanoparticles & laser to treat cancer: An update See pages 6 for balance of the Saturday schedule.

This conference takes place in Eastern Time.

13 Poster Abstracts

PRESENTER ABSTRACT TITLE PAGE Nicolette Harris Administration regimen and feeding program effects on pharmacokinetic and 100 pharmacodynamic profiles of an oral sulforaphane source in beagle dogs Glenna Mauldin Clinical outcome in dogs with apocrine gland adenocarcinoma of the anal sac 101 treated with differing radiotherapy protocols Yasuhiro Fukuyama Use of modified MOHS paste in two cases of multicentric squamous cell 102 carcinoma in situ associated with papillomavirus infection Sergio Vázquez Characterization of tumor associated macrophages in canine lymphomas 103 Atsushi Maeda Outcomes of dogs with localized bile duct carcinoma following surgery: 104 A retrospective study of seven dogs Yusuke Wada Response and outcome of 15 dogs treated with toceranib phosphate with the 105 recurrent nasal carcinoma after radiation therapy Se-hoon Kim Anti-cancer effect of fenbendazole by inducing G2/M arrest and mitotic 106 slippage on canine melanoma cell lines Yoshikawa Ryutaro Intraperitoneal administration of synthetic Microrna-214 exhibits tumor 107 suppression in an intraperitoneal dissemination mouse model of canine hemangiosarcoma Carlos Fonseca-Alves Rapamycin anti-tumoral effect on primary and metastatic canine mammary 108 gland tumor cells in vitro Barbara Borges Cell cycle genes as potential prognostic biomarkers in canine mammary cancer 109 Ryota Iwasaki Identification of sentinel lymph nodes using CT lymphography in dogs with 110 oral malignant melanoma Sita Withers Effect of stimulator of interferon genes (sting) signaling in canine and human 111 osteosarcoma on cytokine/chemokine expression after radiation exposure Miguel de la Virgen Pilot study to evaluate efficay and toxicity for an adjuvant dose-intensified 112 doxorubicin protocol in dogs with hemangiosarcoma following splenectomy Jason Chibuk Owner attitudes toward screening for early cancer detection in pet dogs 113 Takuya Maruo Toxicity evaluation of alternate-day treatment with Teysuno (Tegafur/ 114 Gimeracil/Oteracil) in dog with solid tumor Yuta Nishiyama Re-irradiation after hypofractionated radiotherapy for recurrent intranasal 115 tumors in dogs: A retrospective study Graham Brown Intratumoral treatment of canine high-grade mast cell tumors with Tigilanol 116 Tiglate (EBC-46) Aimee Soileau The effect of arginase on canine lymphocyte function and its modulation by 117 vincristine chemotherapy in dogs with lymphoma Laura Chadsey Treatment of indolent cutaneous T-cell lymphoma (cutaneous lymphocytosis) 118 with hypofractionated radiation therapy in three cats Molly Gasparini Clinical outcome and pathology of canine thyroid carcinosarcoma (2006-2020) 119

14 Poster Abstracts

PRESENTER ABSTRACT TITLE PAGE Allison Gedney Evaluation of the safety and efficacy of coriolus versicolor polysaccharopeptide 120 (i’m yunity®) alone or in combination with doxorubicin for dogs with splenic hemangiosarcoma and its ability to prolong survival Debora Aparecida Pires 3D cell culture – on top method as a reliable precision therapy model in canine 121 de Campos Zuccari mammary tumors William Hendricks Design, analytical validation, and diagnostic yield of a novel canine cancer gene 122 sequencing panel Caitlin Jozwiak Clinical biomarkers of cancer cachexia in cats 123 Aki Ohmi Clinical characteristics and outcomes of lymphoma with mott cell 125 differentiation in miniature dachshunds Gerald Post Safety and toxicity of small molecule inhibitors in dogs with spontaneously 126 occurring malignancies—the use of real world data Wakana Yoneji Certification of heritability of familial gastrointestinal polyposis in Jack Russell 127 terrier and its clinical signs. Matthew Beyers The integrated canine data commons: An open public data sharing platform 128 Jacob Siewert Feline cutaneous lymphoma: a retrospective review of presentation, diagnosis, 129 and response to treatment Kirsten Jackson Analysis of canine myeloid derived suppressor cells (MDSCS) utilizing 130 fluorescence-activated cell sorting, rna protection mediums to yield good quality RNA for single cell analysis using the 10x genomics platform Rothman Reyes Pilot trial of dose-intense combination chop chemotherapy in laboratory dogs 131 Victoria Costa Exploring the association of intratumoral immune cell infiltrates with 132 histopathologic grade in canine mast cell tumors Alex Pyuen Prognostic significance of ki67, agnor, ckit pcr, and kit ihc for completely 133 excised low grade canine mast cell tumors Jacqueline Bowal Prognostic significance of complete response during radiation in feline sino- 134 nasal lymphoma: a retrospective cohort study Lucy Teddy Cyclical 10-day dosing of melphalan for the treatment of canine multiple 135 myeloma Selvi Jegatheeson Response to radioactive iodine (i131) administration in dogs with thyroid 136 carcinoma Luis Lembcke Pharmacokinetics and toxicity of cannadrops™ coconut, a commercially 137 available CBD oil formulation in laboratory dogs Rachel Brady The importance of immunohistochemistry in the diagnosis of suspected non- 138 visceral leiomyosarcoma

15 Sponsors

The Executive Committee of the Veterinary Cancer Society wishes to thank the following companies and organizations for sponsoring portions of the scientific and social programs. Their generosity is passed on to registrants in the form of a lower registration fee.

Platinum Level Gold Level

Silver Level Bronze Level

16 MAKING LIFE BETTER FOR THEM, MAKES LIFE BETTER

VCS looks a little different this year, but opportunities for learning and discovery are still close at hand. That’s why Elanco is proud to sponsor these remote educational sessions.

Grab a virtual front-row seat to these live knowledge shares...

Tanovea®-CA1 Specialty Session (rabacfosadine for injection) and Panel Discussion Frequently Asked Questions Date: Saturday, October 17 Date: Friday, October 16 Time: 4:15-7:15 p.m. EST Time: 10:30 a.m. EST Speakers: Dr. Anne Avery, Dr. Cheryl Speaker: Dr. Doug Thamm London, Dr. Nicola Mason

Can’t make it to these live sessions? Recorded versions will also be available.

Elanco and the diagonal bar are trademarks of Elanco or its affiliates. Tanovea is a registered trademark of VetDC Inc. © 2020 Elanco or its affiliates. PM-US-20-1687

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21 22 JOIN THE VIRBAC TEAM at the VCS 2020 Annual Conference

Get the latest information on mast cell tumor therapy research studies at our Lunch session, Friday, October 16th, 12:15–1:15 PM.

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25 Veterinary Cancer Society for 43 years of service, guiding the profession toward better care and outcomes for patients with cancer.

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30 Friday, October 16, 2020 4:15 pm - 7:15 pm

SPECIALTY SESSION: MAST CELL TUMORS Chaired by: Dr. Cheryl London

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4:15 pm – 5:00 pm The genetic basis of canine mast cell tumors Dr. Heather Gardner 5:05 pm – 5:50 pm Addressing controversies in mast cell tumor surgery Dr. Laura Selmic 5:55 pm – 6:40 pm Medical management of mast cell tumors Dr. Doug Thamm 6:45 pm – 7:15 pm Panel Discussion

Dr. Heather Gardner Dr. Laura Selmic Dr. Doug Thamm

31 THE GENETIC BASIS OF CANINE MAST CELL TUMORS

Presenter: Dr. Heather Gardner

The behavior of canine mast cell tumors is heterogeneous, associated with a variety of clinical and molecular prognostic factors. The contribution of genetic aberrations has long been recognized in the form of activating KIT gene mutations and altered expression of cellular markers of proliferation. More recently, increased efforts charting the genomic landscape of mast cell tumors have provided fundamental insights into both germline and somatic aberrations associated with this disease. Accumulating evidence highlights themes of structural complexity characterized by recurrent mutations and large structural aberrations, that often parallel the presence of KIT mutations and other clinically relevant prognostic features. These genomic findings provide a blueprint for future investigation, however mechanistic studies are warranted to characterize the functional relevance of mutations and optimize clinical translation of these novel genomic studies. The purpose of this seminar is to examine the spectrum of genetic alterations associated with canine mast cell tumors, discuss current limitations in our understanding of canine mast cell tumor genetics, and explore future directions for application of this knowledge.

32 ADDRESSING CONTROVERSIES IN MAST CELL TUMOR SURGERY

Presenter: Dr. Laura Selmic

Mast cell tumor surgery is commonly performed in general and specialty practice. Oncologists and surgeons want to make evidence-based recommendations about the surgery, but there is sometimes a lack of evidence, or that the available evidence is controversial or conflicting. In this wide-ranging lecture, Laura Selmic will highlight some common controversies and present current evidence about these essential topics in mast cell tumor surgery. She will cover surgical dose, surgical margins, pretreatment, lymph node staging with lymphadenectomy, and mast cell tumor histopathology.

One of the most controversial aspects is surgical margins. Traditional surgical recommendations for mast cell tumors have been wide surgical margins, with 3cm lateral margins and one fascial plane deep. Recent evidence will be presented that supports good outcomes in dogs with low-grade tumors with smaller lateral surgical margins. Additionally, available evidence will be discussed about the surgical margins of high-grade tumors. Pretreatment of mast cell tumors prior to surgery is performed to decrease the size of the tumor if present in a difficult location or is large. The shrinkage of the tumor may influence surgical margins taken and the pathology results, which will be discussed. Sentinel lymph node studies are being more commonly performed with subsequent lymphadenectomy of the sentinel lymph nodes performed for staging purposes. The role of this in canine mast cell tumor staging is incompletely defined and will be discussed. The final topic will be mast cell tumor histopathology; reporting has been found to be variable specifically with inconsistent or incomplete reporting of surgical margins, additionally what constitutes incomplete surgical margin may vary between pathologists.

33 MEDICAL MANGAGEMENT OF MAST CELL TUMORS

Presenter: Dr. Douglas Thamm

While surgery remains the mainstay of treatment for canine and feline mast cell tumors (MCTs), there are circumstances where systemic therapy is indicated. These include locally advanced/ disseminated tumors, situations where aggressive local therapy is declined, and adjuvant therapy for tumors at high risk of eventual metastasis. A large number of both traditional cytotoxic and targeted agents have been evaluated for the medical treatment of MCTs; however, there is very little information available regarding efficacy of most treatments when employed postoperatively to delay/prevent local recurrence or metastasis. This lecture will review the data regarding conventional cytotoxic therapies, kinase inhibitors and “frontier” therapies (e.g. monoclonal antibodies, protein kinase C activators) for measurable MCTs in dogs and cats. Combinatorial therapies (e.g. kinase inhibitor/chemotherapy, kinase inhibitor/ radiation therapy combinations) will be reviewed. The (limited) information evaluating postoperative therapy will be discussed, and the available data regarding the utility of specialized testing for treatment allocation (e.g. c-kit gene mutation testing, KIT protein localization) will be discussed.

34 Friday, October 16, 2020 4:15 pm - 7:15 pm

SPECIALTY SESSION: RADIATION ONCOLOGY Chaired by: Dr. Siobhan Haney

Sponsored by

4:15 pm – 5:00 pm Stereotactic body radiation for intra-abdominal tumors Dr. Lyndsay Kubicek

5:05 pm – 5:50 pm Advances in radiotherapy for canine osteosarcoma Dr. Mike Nolan

5:55 pm – 6:40 pm Management of late and rumor associated effects following stereotactic radiation therapy Dr. Susan LaRue

5:45 pm – 7:15 pm Panel Discussion

Dr. Lyndsay Kubicek Dr. Mike Nolan Dr. Susan LaRue

35 STEREOTACTIC BODY RADIATION THERAPY FOR INTRA-ABDOMINAL TUMORS

Presenter: Dr. Lyndsay Kubicek

Stereotactic body radiation therapy (SBRT) involves the precise delivery of highly conformal ablative doses of radiation therapy to targets outside of the brain. This innovative treatment modality was first developed in human therapy as an adaptation of intracranial stereotactic radiosurgery (SRS) and made possible by advances in tumor motion gating and image verification.

Stereotactic radiation has been evaluated in multiple tumor types with central nervous system, nasal and bone tumors being the most studied in veterinary patients. In addition, publications on heart base, injection site sarcomas and adrenal gland tumors have been published in the last 8 years.

To date there is a paucity of literature for stereotactic body radiation to intra-abdominal targets in veterinary medicine. This presentation aims to discuss the role of stereotactic body radiation therapy for intra-abdominal tumors in veterinary medicine through the evaluation of the current literature, case examples and clinical experience, and human radiation therapy protocols. Topics of importance include tumor motion, side effects and possible outcomes.

36 ADVANCES IN RADIOTHERAPY FOR CANINE OSTEOSARCOMA

Presenter: Dr. Mike Nolan

Stereotactic radiotherapy represents an opportunity to advance beyond palliation; it offers an opportunity for effective definitive-intent limb-sparing treatment for extremity osteosarcoma in dogs. However, many questions persist regarding optimal deployment of this powerful therapy: What is the ideal fractionation? How should it be sequenced with chemotherapy? What is the role of bisphosphonates? What defines proper case selection? During this talk, we’ll explore those questions. We’ll also touch on novel approaches that are currently be evaluated in trials, including minimally- invasive limb stabilization strategies, and use of radiation as an anti-metastatic agent for canine osteosarcoma.

37 MANAGEMENT OF LATE AND TUMOR ASSOCIATED EFFECTS FOLLOWING STEREOTACTIC RADIATION THERAPY

Presenter: Dr. Susan LaRue

Stereotactic Radiation Therapy (SRT) has greatly impacted veterinary radiation oncology, decreasing overall treatment time and permitting efficacious treatment of gross disease. SRT provides radiation alternatives for tumors in previously inaccessible locations. As with traditional fractionated radiation, the concept uncomplicated tumor control should remain a fundamental principle of SRT. This principle is based on the concept that a certain percent of patients may be more suspectable to clinically significant late effects. However, lessening the dose to minimize late effects may underdose the rest of the patient population, reducing tumor control and resulting in decreased overall survival. In addition to late radiation effects, SRT seems to have a more profound impact on gross disease that can lead to complications requiring management.

This talk will address prevention of radiation effects by proper adherence to important normal tissue constraints, and guidelines for providing standardized contouring information. It will also address identification of important pretreatment, intra treatment and post treatment factors that may adversely impact outcome as well as drugs used during and after treatment that may help mitigate radiation effects. Included will be guidance of how to determine when and what type of surgical management is needed when late radiation effects occur following SRT.

Some tumors, because of size and location, may not have surgical options, and tumor control following fractionated radiation may be historically poor. Adverse effects following SRT can occur when tumors that have already invaded and destroyed adjacent bone and other tissues. For these cases, consultation with a surgical oncologist is necessary prior to SRT.

SRT is an important tool in veterinary oncology. Radiation oncologists need to continually re-evaluate tumor control and radiation effects in their patients to improve therapeutic gain and optimize protocols. Surgical oncologists are important team members and should work with radiation oncologists to improve management of late effects.

38 Friday, October 16, 2020 Thursday, October 15, 2020 4:15 pm - 7:15 pm

SPECIALTY SESSION: IMMUNOTHERAPY Chaired by: Dr. Sita Withers

Sponsored by

4:15 pm – 5:00 pm Immunotherapy strategies for “heating up” the tumor microenvironment Dr. Sita Withers

5:05 pm – 5:50 pm New strategies to stimulate effective tumor immunity by modifying the tumor microenvironment Dr. Stephen Dow

5:55 pm – 6:40 pm Targeted radionuclide therapy and the interface between radiation and immunotherapy Dr. David Vail

6:45 pm – 7:15 pm Panel Discussion

Dr. Sita Withers Dr. Stephen Dow Dr. David Vail

39 IMMUNOTHERAPY STRATEGIES FOR “HEATING UP” THE TUMOR MICROENVIRONMENT

Presenter: Dr. Sita Withers

Adaptive T lymphocyte immunity against tumor neoantigens is the goal of many cancer immunotherapies. Components of the innate immune system are required to facilitate this response, but can also hinder immunotherapeutic strategies. Consequently, there is increasing evidence that the efficacy of therapeutics promoting T lymphocyte effector functions can be enhanced by concurrent modulation of innate immune cell functions. For example, some therapeutic strategies that target innate immune cell functions include: the promotion of dendritic cell recruitment and activation; depletion or inhibition of myeloid-derived suppressor cells (MDSCs) and M2- type macrophages; improvement in the pro-inflammatory cytokine and chemokine milieu; and suppression of inhibitory cytokines and enzymes. Modulation of innate immune system components in this way can help create an immunologically “hot” tumor microenvironment that facilitates T lymphocyte maturation, from antigen priming to effector function.

Ongoing studies by our group suggest that some osteosarcoma cells are defective in their ability to induce inflammation in response to genomic instability and DNA damage. This deficiency arises due to decreased expression of stimulator of interferon genes (STING) in some cell lines. STING typically functions to link the detection of cytoplasmic DNA with induction of type I interferon and chemokine expression, which is critical for recruitment and activation of effector cells. While STING signaling appears crucial for the development of radiation-induced systemic anti-cancer immunity (i.e. the abscopal effect) in some models, deficiencies in STING expression also predispose cancer cells to enhanced susceptibility to oncolytic DNA viruses. Therefore, understanding how tumor cells dampen or promote components of the host innate immune system will improve our ability to create rational immunotherapeutic combinations.

40 NEW STRATEGIES TO STIMULATE EFFECTIVE TUMOR IMMUNITY BY MODIFYING THE TUMOR MICROENVIRONMENT

Presenter: Dr. Steven Dow

Cancer immunotherapy: Current Challenges: The promise of cancer immunotherapy has now been rapidly realized, in studies largely conducted just over the past decade. Despite tremendous advances, challenges remain, particularly with respect to treatment of immunologically “cold” tumors such as pediatric sarcomas and malignant gliomas. Solid tumors in general represent a significant barrier to adoptive T cell transfer, and checkpoint antibody therapeutics are generally relatively ineffective in low T cell infiltrated cancers. Personalized cancer vaccines are difficult currently to produce quickly, and induction of effective immunity is not always possible.

Role of the TME in regulating tumor immunity. The immune suppressive tumor microenvironment (TME) is a major impediment to effective cancer immunotherapy. Particular TME barriers include immune suppressive stromal cells (eg, tumor macrophages, cancer fibroblasts, Tregs) and physical stromal barriers such as fibrotic responses and extracellular matrix. Overcoming these local tumor barriers represents a fundamental challenge to successful cancer immunotherapy.

Modifying the TME. One approach to reduce immune suppression within the TME is to deplete or functionally alter immune suppressive cell populations. We have evaluated several different strategies. In one approach, macrophages can be depleted directly (eg, liposomal clodronate) or activated locally (eg, by injection of TLR3 or 9 agonists). A third strategy, which we have been investigating in both osteosarcoma and glioma trials, is to induce sustained blockade of recruitment of inflammatory monocytes to the tumor, which over time leads to macrophage depletion. To accomplish this clinically in dogs, we have repurposed the angiotensin receptor blocker (losartan) as a novel CCR2 antagonist and monocyte migration inhibitor, together with toceranib, which leads to Treg depletion. The two drugs in combination have been shown to induce tumor regression in metastatic osteosarcoma and to enhance vaccine effectiveness in glioma. Additional studies are underway to more fully define the mechanisms of action of these drugs in canine cancer.

41 TARGETED RADIONUCLIDE THERAPY AND THE INTERFACE BETWEEN RADIATION AND IMMUNOTHERAPY

Presenter: Dr. David M. Vail

In situ tumor vaccination is a therapeutic strategy that seeks to convert a patient’s own tumor into a nidus for presentation of tumor-specific antigens in a way that will stimulate and diversify an anti-tumor T cell response. Radiation therapy (RT) may serve as a method of in situ tumor vaccination. The mechanisms whereby RT interacts with tumor cells and the TME include: 1) temporary local eradication of RT-sensitive immune lineages including suppressor and effector lymphocytes; 2) local release of inflammatory cytokines and damage-associated molecular patterns resulting in local effects on endothelial cell expression of adhesion receptors, immune cell trafficking, and immune cell activation; 3) immunogenic tumor cell death and release of tumor-specific antigens; and 4) induction of phenotypic changes in the expression of immune susceptibility markers on tumor cells surviving RT. Consequently, RT enhances dendritic cell maturation, antigen cross-presentation, and diversification of anti-tumor T cell response. However, the capacity of external beam RT (EBRT) targeting one tumor to elicit a systemic anti-tumor immune response is limited. This occurs in part, from the local and systemic effects of immunosuppressive cells that pervade the TME in distant non-radiated tumors. Although EBRT may prime a more effective T cell response, when these effector T cells (Teff) circulate to non-radiated tumors they encounter a suppressive TME and poorly susceptible tumor cells. Furthermore, suppressive immune cells [e.g. regulatory T cell (Tregs)] in distant non-radiated metastatic tumors may circulate to the radiated tumor site after RT, and inhibit local priming of tumor reactive T cells. This reflects a need to further address the suppressive TME of distant tumors not targeted by EBRT. While it is not typically feasible to deliver EBRT to all sites of metastatic disease (due to immune suppression and inability to target microscopic disease), we can achieve this using molecular targeted radiotherapy (MTRT).

42 Saturday, October 17, 2020 4:15 pm - 7:30 pm

SPECIALTY SESSION: LYMPHOMA Chaired by: Dr. Nicola Mason

Sponsored by

4:15 pm – 5:00 pm Molecular and biological heterogeneity of canine lymphoma Dr. Anne Avery

5:05 pm – 5:50 pm Enhancing the efficacy of immunotherapy in diffuse large B cell lymphoma (DLBCL) using rational combination approaches Dr. Cheryl London

5:55 pm – 6:55 pm Adaptive T cell therapies for the treatment of lymphoma Dr. Nicola Mason

7:00 pm – 7:30 pm Panel Discussion

Dr. Anne Avery Dr. Cheryl London Dr. Nicola Mason

43 MOLECULAR AND BIOLOGICAL HETEROGENEITY OF CANINE LYMPHOMA

Presenter: Dr. Anne Avery

Lymphoproliferative disorders can be broadly classified into immature neoplasms (acute leukemia), mature B cell tumors and mature T cell tumors. Within these groups are an array of subtypes that arise from cells in different stages of development and with different functions. For example, CD4+ peripheral T cell lymphoma in dogs arises from a T cell that has not yet been activated by antigen and is recently emigrated from the thymus. By contrast, T zone lymphoma appears to be derived from a previously activated T cell and has a transcriptome most closely associated with effector T cells. In people, diffuse large B cell lymphoma can be subdivided into two clinically discrete subtypes by transcriptome profiling, and four clinically discrete subtypes by mutational analysis – each subtype is thought to arise from B cells in different phases of the germinal center/post germinal center reaction. Although most canine and human lymphomas are currently treated with broadly active cytotoxic therapies, as our understanding of the unique pathways, mutational features and antigen expression in different subtypes increases, the possibility of using more targeted therapies becomes more realistic. Both dogs and people can benefit from the use of dogs as a pre-clinical model for experimental therapies, and therefore it is important to identify where human and canine subtypes are similar and where they differ. In this session, classification, dysregulated pathways and the cell of origin of canine lymphoma/leukemia subtypes will be discussed, with an emphasis on similarities and differences with their human counterparts.

44 ENHANCING THE EFFICACY OF IMMUNOTHERAPY IN DIFFUSE LARGE B CELL LYMPHOMA (DLBCL) USING RATIONAL COMBINATION APPROACHES

Presenter: Dr. Cheryl London

Authors: London CA1, Hendricks WPD2, Avery AC3, Wood CA1, Gardner HL1, Richards KL4 1Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA; 2Translational Genomics Research Institute, Phoenix, AZ; 3College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO; 4College of Veterinary Medicine and Weill College of Medicine, Cornell University, Ithaca, NY.

Despite the addition of rituximab to CHOP chemotherapy in the treatment of diffuse large B cell lymphoma (DLBCL), up to 40% of human patients ultimately succumb to their disease. Additionally, those patients effectively cured have a high risk of long-term morbidities secondary to treatment. As such, novel approaches that reduce the intensity of chemotherapy while simultaneously improving outcomes through combination with small molecule inhibitors/immunotherapeutics are desirable. Unfortunately, exploration of such treatment strategies is typically relegated to patients with relapsed/refractory disease where efficacy is likely to be lower. It is now clear that DLBCL in dogs resembles the human disease with respect to clinical presentation, molecular aberrations, and genomic alterations making it a good spontaneous large animal model of disease. As such, we have been leveraging canine DLBCL to rapidly evaluate the activity of rational small molecule inhibitors targeting PI3Kδ (RV1001), NAMPT/PAK4 (KPT- 9274), or SUMO-Activating Enzyme (TAK-981) combined with immunotherapy (anti-CD20, from Elanco), with the ultimate goal of identifying the most effective combination to move forward in human patients. Using an adaptive mini-pilot trial approach in the front-line setting we are assessing the activity and toxicities of anti-CD20/targeted small molecule combinations in affected dogs and correlating clinical, biological, and genomic biomarkers to develop signatures that predict response to therapy and long-term progression-free survival. Our data demonstrate that anti- CD20 effectively depletes B cell populations in treated dogs and that treatment with RV1001 can break tolerance as evidenced by prednisone responsive transaminitis. We have identified long-term survivors in each treatment cohort, and our preliminary data suggest that a combination of low dose doxorubicin, anti-CD20, KPT-9274 and RV1001 may be associated with the long-term survival. We have performed integrated genomic analysis of paired tumor/ normal and matched constitutional samples from the 5 dogs in first cohort to identify candidate molecular correlates of treatment response. Matched tumor/normal whole genome sequencing confirmed previously described (TRAF3, SETD2, TP53) as well as novel candidate (MGA) driver mutations associated with canine DLBCL. Following completion of our pilot studies and correlative analyses, a large prospective study will be undertaken to confirm findings.

45 ADAPTIVE T CELL THERAPIES FOR THE TREATMENT OF LYMPHOMA

Presenter: Dr. Nicola Mason

T cells that exhibit specificity towards tumor associated antigens represent a powerful natural tool that can be manipulated to enhance their therapeutic effects against both solid and hematological malignancies. Frequently, these manipulations occur ex-vivo and involve expanding either peripheral T cells or those extracted from the tumor itself (tumor infiltrating lymphocytes or TILs) to generate large numbers of tumor specific T cells that are then adoptively transferred back into the patient. Several strategies have been employed to increase the percentage of tumor-specific T cells prior to ex vivo expansion. These include vaccination of the patient prior to T cell harvest and genetic modification of T cells to express a synthetic T cell receptor or chimeric antigen receptor (CAR) specific for the tumor antigen of interest after T cell harvest. Both methodologies are currently being tested in clinical trials in the veterinary clinics. In the human arena, perhaps the most remarkable clinical results to date have been achieved using autologous CAR-T cells in patients with hematological malignancies such as acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL), B-Non Hodgkin Lymphoma (B-NHL) and multiple myeloma (MM). In dramatic fashion, adoptively transferred CAR-T cells have been shown to rapidly and effectively eliminate kilograms of tumor and provide durable remissions that correlate with long-term persistence of these cells. Next- generation CAR-T strategies are now focused on developing allogeneic, off-the-shelf products that can function within the immunosuppressive microenvironment, which aims to improve CAR-T effectiveness in solid tumors. The tools and protocols to generate autologous and allogeneic tumor-specific T cells are now available in the veterinary field and clinical trials are underway to assess their safety and efficacy in dogs with B cell lymphoma. In this lecture we will explore the current use and up-coming strategies to improve upon current adoptive T cell therapies in dogs with B cell lymphoma, outlining how the field is moving towards obtaining durable clinical responses in dogs with this challenging disease.

46 Saturday, October 17, 2020 4:15 pm - 7:15 pm

SPECIALTY SESSION: SURGICAL ONCOLOGY Chaired by: Dr. Will Eward

Sponsored by

4:15 pm – 5:00 pm The orthopedic oncologist: The butcher, the baker AND the candlestick maker Dr. Julia Visgauss

5:05 pm – 5:50 pm Application of 3D printing technology in maxillofacial oncologic surgery Dr. Marine Traverson

5:55 pm – 6:40 pm Single-session cryoblation for local tumor control and pain relief: Evidence and case review Dr. Alan Sag

6:45 pm – 7:15 pm Panel Discussion

Dr. Julia Visgauss Dr. Marine Traverson Dr. Alan Sag

47 THE ORTHOPEDIC ONCOLOGIST: THE BUTCHER, THE BAKER, AND THE CANDLESTICK MAKER

Presenter: Dr. Julia Visgauss

As knowledge and technology advance, the practice of medicine gets more and more specialized. This is especially true of practices based in large academic tertiarily referral centers, and physicians who care for patients with rare diseases. Furthermore, within orthopedic surgery, general practice is rare, and we are even seeing specialization within subspecialty disciplines. However, the practice of orthopedic oncology offers a unique dichotomy. While we provide highly specialized care for patients with a rare cancer, our scope of practice is quite diverse; not only with regard to anatomic location of tumors, but also with a relevant understanding of radiology and pathology that allows us to provide safe and efficient care to our patients.

We interpret our own musculoskeletal imaging with regard to differential diagnosis, and need for biopsy. We are often required to interpret chest imaging of patients seen in clinic for sarcoma surveillance prior to availability of an official radiology report. We participate in the interpretation of intraoperative biopsies that affect surgical decision making, which may be done with a non-musculoskeletal pathologist. Furthermore, even at locations where a multidisciplinary sarcoma team exists- the orthopedic oncologist has the unique perspective of the patient’s clinical presentation in its entirety. Thus, it is important to be able to interpret all of the data in order to make the most appropriate clinical decisions. Here I present a few challenging cases that highlight the importance of this comprehensive base of knowledge, and how it directly impacted the care and outcome of these patients.

48 APPLICATIONS OF 3D PRINTING TECHNOLOGY IN MAXILLOFACIAL ONCOLOGIC SURGERY

Presenter: Dr. Marine Traverson

Computer-aided design and manufacturing, and 3-dimensional (3D) printing have increased workflow efficiency and accuracy in human medicine, and find applications in neurologic, orthopedic, maxillofacial and oncologic surgery via the production of pre-surgical visualization models, cutting and drilling guides, and custom-made implants. The use of this technology is nascent in veterinary medicine, and offers the ability to produce individual custom-made biomedical devices and implants to reconstruct complex anatomic defects and restore function in oncologic surgery. This presentation will review different prototypes, preclinical and clinical cases using 3D printing and computer aided design and manufacturing as a novel planning and reconstructive approach to canine maxillofacial oncologic surgery. Some examples discussed will include among other things the use of a cutting and drilling guide to improve margin accuracy, surgical time, workflow and protection of the surrounding soft tissues in caudal maxillectomy, and a case of dorsal maxillary multilobular osteochondrosarcoma treated with 3D printed custom-made devices.

49 SINGLE-SESSION CRYOABLATION FOR LOCAL TUMOR CONTROL AND PAIN RELIEF: EVIDENCE AND CASE REVIEW

Presenter: Alan Alper Sag, MD – Duke University Medical Center

Cryoablation is now mainstream in the treatment of tumors and tumor-associated pain. The procedure is ideally performed using computed tomography and ultrasound guidance. Treatments can include tumors of bone, liver, kidney, lung, and soft tissues, and can be used to freeze nerves for pain relief. In this presentation we will discuss the utility and technical details for how to perform a cryoablation, with specific focus on safety and avoidance of complications.

50 Saturday, October 17, 2020 4:15 pm - 7:25 pm

SPECIALTY SESSION: ALTERNATIVE THERAPIES & PALLIATIVE CARE Chaired by: Dr. Shea Cox

4:15 pm – 4:45 pm Philosophy of Hospice and Palliative Care Dr. Christine Culler

4:50 pm – 6:20 pm In-home Palliative Care Management: What can be provided and how it can help Dr. Shea Cox

6:25 pm – 7:25 pm Euthanasia and Natural Death Dr. Page Yaxley

Dr. Shea Cox Dr. Christine Culler Dr. Page Yaxley

51 PHILOSOPHY OF HOSPICE AND PALLIATIVE CARE

Presenters: Christine Culler, DVM, MS, DACVECC

There is a common misperception that hospice = imminent death, which is far from the truth. During this 30-minute introductory session, the presenters will discuss hospice and palliative care concepts with a special focus on how palliative care can augment the care provided by the oncology team. An interactive discussion around the personal challenges faced by the audience as end of life nears will help guide context for the following sessions.

52 IN-HOME PALLIATIVE CARE MANAGEMENT: WHAT CAN BE PROVIDED AND HOW IT CAN HELP

Presenters: Shea Cox, DVM, CHPV, CVPP

When traditional therapies fail, what more can we do to provide comfort and quality of life? This is a question asked by many oncology teams, and this session will share the many palliative options and approaches to care that are available for end-stage disease management from a hospice lens. Case studies will be presented to highlight successful in-home, client-administered care, including the importance and utilization of “Comfort Kits” to aid pets and their families during crisis episodes.

53 EUTHANASIA AND NATURAL DEATH

Presenters: Page Yaxley, DVM, DACVECC

This session will discuss topics pertaining to euthanasia and natural death, including tips to provide a good death in a clinic setting, pre-euthanasia sedation protocols for various situations and species, alternative AVMA-approved euthanasia techniques when venous access is difficult or not possible, how to navigate a hospice-assisted natural death when euthanasia is not an option, and client communication tips.

54 Friday, October 16, 2020

Resident Oral Abstracts

55 SAFETY EVALUATION OF LOW DOSE MELOXICAM WITH TOCERANIB PHOSPHATE (PALLADIA) IN CANCER BEARING CATS: A PHASE I DOSE-FINDING STUDY

Presenter Name: Samuel Keepman Presenter Institution: University of Wisconsin Presenter Email: [email protected]

Authors: Keepman, Samuel1; Pellin, MacKenzie1 1University of Wisconsin, Madison, WI

Introduction: Palladia is used to treat several feline cancers. NSAIDs are infrequently utilized in cats due to concern for renal compromise; however, recent studies demonstrate tolerability of low dose meloxicam. The goal of this study was to determine the maximum tolerated dose and adverse event profile of combined Palladia and meloxicam in cancer bearing cats. Secondary goals were to assess anti-cancer tumor efficacy and impact upon quality of life and analgesia.

Methods: Cats with any cancer not involving the kidneys were eligible. The study adopted a conventional 3+3 cohort design. Palladia was administered every other day at standard dose with meloxicam administered in an escalating fashion, at a starting dose of 0.01mg/kg on days opposite of Palladia. Lab work, blood pressure, tumor measurements, pain score, and client-completed quality of life surveys were recorded every 2-4 weeks during the 3 month study period.

Results: Fifteen cats have been enrolled in the study to date. The MTD of meloxicam when combined with Palladia has not yet been reached, but it is at least 0.01mg/kg every other day with no cats being withdrawn due to adverse events from the drug combination. The majority of cats have demonstrated clinical benefit with stable to mildly improved tumor measurements, quality of life, and pain scores.

Conclusion: Preliminary data indicate that low dose meloxicam combined with Palladia is safe and well-tolerated in cancer-bearing cats. Continued patient recruitment and data collection is needed to determine the maximum tolerated dose of meloxicam. The results of our study will guide further Phase II/III trials.

Funding Information: Funding for this study has been provided by the VCS Resident Grant.

56 LOMUSTINE BASED CHEMOTHERAPY PROTOCOLS FOR THE TREATMENT OF CANINE T-CELL LYMPHOMA

Presenter Name: Gabrielle Carter Presenter Institution: VCA California Veterinary Specialists Presenter Email: [email protected]

Authors: Carter, Gabrielle1; Ogilvie, Gregory1; Diniz, Pedro2; Elias, Lourdes1; Proulx, David1 1VCA California Veterinary Specialists 2Western University of Health Sciences, Pomona, CA

Introduction: Dogs diagnosed with T-cell lymphoma (TCL) generally have a poor prognosis even with the use of multi-agent chemotherapy protocols. Lomustine has shown promise when used to treat TCL, however it is unclear if a multi-agent protocol utilizing lomustine is superior to standard CHOP chemotherapy. Our objective is to determine the prognosis and associated prognostic factors for dogs that were diagnosed with TCL, via flow cytometry, and treated with either a modified CHOP chemotherapy protocol (including lomustine) or a single agent lomustine protocol.

Methods: Retrospective medical record review of one hundred and nine dogs diagnosed with TCL from May 2010 to December 2019. Data extracted from medical records included signalment, weight, location of disease, stage, substage, anemia, neutropenia, monocytopenia, chemotherapy protocol, completion of chemotherapy protocol, recurrence, date of death, and cause of death. Dogs were separated into two cohorts based on chemotherapy protocol. Ninety-five dogs received a modified CHOP protocol where lomustine was substituted for cyclophosphamide. Fourteen dogs received a single agent lomustine protocol.

Results: Forty patients completed their chemotherapy protocol, thirty-five in the modified CHOP group and five in the single agent lomustine group. Of these, twenty-nine patients had documented recurrence with a median disease- free interval (DFI) of 263 days. Additional data to be presented.

Conclusion: Although TCL continues to be a difficult disease to treat, multi-agent protocols utilizing lomustine provide reasonable DFIs and mean survival times (MST). As expected, dogs receiving multi-agent protocols experienced longer MST than dogs receiving single agent lomustine.

57 OUTCOME IN 27 DOGS WITH CURATIVE-INTENT TREATMENT OF LOCALIZED PRIMARY PULMONARY HISTIOCYTIC SARCOMA

Presenter Name: Caroline Murray Presenter Institution: University of California, Davis Presenter Email: [email protected]

Authors: Murray, Caroline1; Willcox, Jennifer1; De Mello Souza, Carlos2; Husbands, Brian3; Cook, Matthew3; Clifford, Craig4; Leeper, Haley5; Pellin, MacKenzie6; Richardson, Danielle7; Herrera, Chamisa8; Krick, Erika9; McMillan, Sarah10; Al-Nadaf, Sami1; Skorupski, Katherine1 1University of California, Davis, CA 2University of Florida, Gainesville, FL 3The Ohio State University, Columbus, OH 4Hope Veterinary Specialists, Malvern, PA 5Oregon State University, Corvallis, OR 6University of Wisconsin-Madison, Madison, WI 7Ontario Veterinary College, Guelph, ON, Canada 8Boundary Bay Veterinary Specialty Hospital, Vancouver, BC, Canada 9Mount Laurel Animal Hospital, Mount Laurel, NJ 10Veterinary Emergency Referral Center of Hawaii, Honolulu, HI

Introduction: Primary pulmonary histiocytic sarcoma (PHS) is a rare form of dendritic cell neoplasia originating within the pulmonary parenchyma. There is limited literature describing prognosis in dogs with PHS receiving curative-intent treatment consisting of surgical excision and adjuvant systemic chemotherapy. The primary objective of this study was to gain insight on localized primary PHS and provide information on outcome with standardized local and systemic therapy. A secondary objective was to identify prognostic factors in this population.

Methods: A multi-institutional study was performed and medical records were retrospectively reviewed. For inclusion, dogs were required to have confirmed localized PHS and they must have undergone curative-intent surgery with resection of all gross primary tumor and involved lymph nodes; additionally, they must have received treatment with adjuvant single-agent CCNU chemotherapy.

Results: Twenty-seven dogs from 11 institutions were included in analyses. The overall median survival time (MST) was 432 days. Only CCNU dose was found to be associated with survival in this population. The MST for dogs prescribed less than the median dose (66.6 mg/m2) was 673 days vs 200.5 days for dogs prescribed greater than or equal to 66.6 mg/m2 (p=0.005). Factors that were not found to be associated with survival included body weight, breed, symptoms at diagnosis, hypoalbuminemia, tumor size, lung location, lymph node metastasis, surgical margins, and CCNU dose reductions.

Conclusion: Dogs diagnosed with localized PHS and treated with curative-intent surgery and adjuvant CCNU chemotherapy have a relatively favorable prognosis and aggressive treatment may be advisable to attempt to prolong survival.

Funding Information: N/A

58 FIRST IN PATIENT TRIAL OF HIGH INTENSITY FOCUSED ULTRASOUND (HIFU) FOR TREATMENT OF CANINE SOLID TUMORS.

Presenter Name: Jennifer Carroll Presenter Institution: Virginia-Maryland College of Veterinary Medicine Presenter Email: [email protected]

Authors: Carroll, Jennifer1; Coutermarsh-Ott, Sheryl1; Tuohy, Joanne1; Barry, Sabrina1; Klahn, Shawna1; Allen, Irving1; Ruth, Jeffery2; Dervisis, Nick1 1Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA 2IDEXX, Westbrook, Maine

Introduction: High Intensity Focused Ultrasound (HIFU) is a non-invasive tissue ablative technique approved for specific benign tumors in humans. Preclinical modeling suggests that HIFU may induce systemic antineoplastic immune responses. We designed, executed, and report the results of a pilot trial of HIFU for dogs diagnosed with subcutaneous solid tumors. Our primary objective was to evaluate the safety and feasibility of delivering HIFU to canine cancer patients. Our secondary objectives were to characterize the immediate adverse events, the pathologic changes in the treated tumor tissue, and the changes to the treated tumor microenvironment.

Methods: Dogs with subcutaneous solid tumors were recruited. Pretreatment biopsies were obtained and a single HIFU treatment for partial tumor ablation was delivered. Tumors were excised 4-6 days post HIFU and submitted for histopathology and immunohistochemistry. Gene expression analysis using a custom superarray was used to evaluate immunologic changes in the intratumoral microenvironment.

Results: 20 dogs were recruited and treated. Tumors histologies included 15 soft tissue sarcomas, 3 mast cell tumors, 1 osteosarcoma, and 1 thyroid carcinoma. Complications secondary to HIFU were generally self-limiting and included various degrees of cutaneous thermal injury. Immunohistochemistry revealed an increase in the number of T-cells within the periphery of the ablation zone. Quantitative RT-PCR revealed a > 2 fold increase in genes associated with pro-inflammatory cytokine signaling in post treatment samples.

Conclusion: HIFU appears to be feasible, safe, and results in predictable tumor ablation characterized by discrete regions of coagulative necrosis. HIFU resulted in tentative immunostimulatory alterations to the tumor microenvironment.

Funding Information: Focused Ultrasound Foundation and Theraclion

59 A MULTIMODALITY RADIOIMMUNOTHERAPY APPROACH TO TREATING ADVANCED STAGE CANCER IN COMPANION DOGS.

Presenter Name: Kara Magee Presenter Institution: University of Wisconsin Presenter Email: [email protected]

Authors: Magee, Kara1; Hernandez, Reinier1; Turek, Michelle1; Marsh, Ian1; Bednarz, Bryan1; Grudzinski, Joseph1; Weichert, Jamey1; Sondel, Paul1; Albertini, Mark1; Morris, Zachary1; Vail, David1 1University of Wisconsin, Madison, Wisconsin

Introduction: Following immunotherapy, a minority of advanced stage cancer patients develop immune responses resulting in durable tumor regression despite widespread metastasis. To expand this minority, we investigate external beam radiation (EBRT) and an intra-tumoral immunocytokine to create an in situ vaccine in one tumor microenvironment and combine this approach with systemic targeted radionuclide therapy (TRT) in order to propagate an abscopal effect at metastatic sites. We have documented durable responses in murine models and now present bridging data in companion dogs.

Methods: Five dogs with advanced stage melanoma or osteosarcoma were included. A tumor-targeting alkylphosphocholine (NM600) that chelates radionuclides was used as a theranostic for serial diagnostic 86Y- NM600 PET/CT imaging of the primary tumor and metastatic sites. These images enabled Monte Carlo dosimetry calculations for subsequent delivery of therapeutic 90Y-NM600 to all sites. Concurrently, 8 Gy EBRT was delivered to the primary tumor followed by 3 daily intratumoral injections of hu14.18-IL2 immunocytokine. Adverse event (AE) data and biospecimens were collected.

Results: All metastatic tumors had differential uptake of 86Y-NM600 as evidenced by PET/CT and a minimum immunomodulatory dose of 2 Gy 90Y-NM600 was successfully delivered to all metastatic sites in 4 of 5 dogs. Treatments were well tolerated and AEs were transient/low grade.

Conclusion: We confirmed tumor-selective uptake and the theranostic potential of NM600 to stage, create subject- specific dosimetry, and safely deliver TRT to metastatic sites in dogs with osteosarcoma and melanoma. Ongoing investigations include characterizing immunomodulatory and anti-tumor responses in larger cohorts along with investigations of higher TRT dosing and alternative radionuclides.

Funding Information: Funded in part by the following: U01CA233102-01 DHHS, PHS, NIH and The Barbara A. Suran Comparative Oncology Research Institute.

60 SAFETY AND FEASIBILITY OF WHOLE LUNG IRRADIATION IN THE TREATMENT OF CANINE APPENDICULAR OSTEOSARCOMA

Presenter Name: Amanda Brehm Presenter Institution: Texas A&M University Presenter Email: [email protected]

Authors: Brehm, Amanda1; Wilson-Robles, Heather1; Miller, Tasha1; Jarvis, Jill1; Deveau, Michael1 1Texas A&M University

Introduction: Recent attempts to improve the long-term prognosis for canine osteosarcoma with intensified chemotherapy regimens have largely been unsuccessful. Whole lung irradiation (WLI) has been used successfully in humans as an adjuvant treatment for osteosarcoma. The aim of this study is to describe the safety and feasibility of WLI in dogs with appendicular osteosarcoma. Our hypothesis is that WLI as an adjuvant to standard of care treatment will be safe and well tolerated.

Methods: Twelve client-owned dogs with appendicular osteosarcoma were enrolled in this prospective clinical trial. Dogs were included if they had completed amputation and 4 doses of carboplatin without radiographic evidence of metastasis. Ten once-daily fractions of 1.75 Gray were administered to 100% of the lung volume, beginning 3 weeks after the last dose of carboplatin. Dogs were monitored with physical examinations and chest radiographs every 8 weeks after WLI.

Results: The most common side effect during radiation therapy was weight loss (n = 8), with an average loss of 4% body weight per dog. Hematologic toxicity occurred in 6 dogs and was low grade in all cases. No dogs developed overt pneumonitis. The median follow-up time following radiation therapy was 20 weeks. No dogs experienced late side effects attributable to radiation therapy. No pathologic changes were identified on thoracic radiographs prior to disease progression in any dog.

Conclusion: WLI appears to be safe and well tolerated in dogs. This protocol warrants further investigation for toxicity characterization and efficacy evaluation with a larger scale clinical trial.

Funding Information: GINN Grant (Internal funding through Texas A&M) ACVIM Resident Research Grant Katherine and Rebecca Rochelle Chair in Oncology

61 PROTEASOME INHIBITION VIA BORTEZOMIB INDUCES APOPTOSIS IN CANINE GLIOMA CELLS

Presenter Name: Brittany Feldhaeusser Presenter Institution: University of Georgia Presenter Email: [email protected]

Authors: Feldhaeusser, Brittany1; Nagata, Koichi1; Gogal, Robert1; Laver, Travis1 1University of Georgia

Introduction: Gliomas are the second most common canine primary brain tumor. These tumors are most commonly treated with radiation therapy and generally have a guarded prognosis. The role of systemic therapy in treating canine gliomas is largely unresolved; However, recent human glioma data suggest that bortezomib (BORT) (Velcade®), a proteasome inhibitor, in combination with radiation therapy may lead to an improved clinical outcome.

Methods: We identified the IC50 of BORT in two canine glioma cell lines, SDT3G and J3TBG, using an ATP-based cell viability assay. We then treated cells with two doses of BORT (50 nM and 75 nM) and assessed the effects of this treatment on apoptosis via flow cytometry.

Results: Treatment of canine glioma cells resulted in a dose-dependent decrease in cell-viability with IC50 values calculated as 45 nM (J3TBG) and 67 nM (SDT3G).

Treatment of the J3TBG cell line with BORT resulted in a decreased percentage of viable cells and an increased percentage of apoptotic cells at both 48 and 72 hr. Both of these effects were statistically significant in comparison to the vehicle only (DMSO) treated cells (p < 0.01). Furthermore, there was a statistically significant increase in apoptosis between the 50 nM and 75 nM doses, indicating a dose-dependent response. Similar responses were seen in the SDT3G cell line.

Experiments to assess the effect of radiation plus BORT on the clonogenic potential of canine glioma cells are ongoing.

Conclusion: BORT significantly reduces the viability of canine glioma cells via induction of apoptosis.

Funding Information: This work was funded by a Resident Research Grant from the Veterinary Cancer Society.

62 CHARACTERIZING CIRCULATING NUCLEOSOMES IN THE PLASMA OF DOGS WITH LYMPHOMA

Presenter Name: Christopher Dolan Presenter Institution: Texas A&M University Presenter Email: [email protected]

Authors: Dolan, Christopher1; Wilson-Robles, Heather1; Miller, Tasha1; Jarvis, Jill1; Terrell, Jason2; Dewsbury, Nathan3; Herzog, Marielle4; Kelly, Terry2; Bygott, Thomas4; Michel, Gaetan4 1Texas A&M University 2Volition America 3Volition Veterinary Diagnostic Development 4Belgian Volition

Introduction: Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death which causes chromatin fragmentation and release of nucleosomes into the blood. The Nu.QTM platform exploits the different compositions of circulating nucleosomes in the blood of humans that occurs with disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of lymphoma (LSA) using the Nu.QTM platform of assays.

Methods: A total of 70 dogs diagnosed with LSA and 65 healthy controls were recruited for this study. The LSA samples were recruited from TAMU or purchased from various biobanks. All control samples were recruited from TAMU. Graphpad Prism v.8 was used to make comparisons between LSA and controls and within the LSA cohort.

Results: Dogs with LSA had an approximately 10-fold increase in their plasma nucleosome concentrations compared to controls (AUC 84.6%). Nucleosome concentrations increased with the stage of the disease.

Conclusion: The Nu.QTM platform was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes may be useful for differentiating cancer from healthy individuals in canines. Further testing is underway to better characterize LSA and optimize the Nu.QTM platform for canine LSA detection.

Funding Information: Salary for Mrs. Tasha Miller was provided by the Fred and Vola Palmer Chair for Comparative Oncology held by Dr. Wilson-Robles.

63 PD-1 AND PD-L1 EXPRESSION ON CANINE UROTHELIAL CARCINOMA CELL LINES AND THE RELATIVE EXPRESSION OF LYMPHOCYTE PD-1 IN CANINE UROTHELIAL CARCINOMA PATIENTS

Presenter Name: Christopher Pinard Presenter Institution: Ontario Veterinary College, University of Guelph Presenter Email: [email protected]

Authors: Pinard, Christopher1; Stegelmeier, Ashley2; Inkol, Jordon3; Poon, Andrew3; Mutsaers, Anthony4; Wood, Geoffrey2; Wood, Darren2; Woods, J Paul1; Hocker, Samuel5 1Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 2Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 3Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 4Departments of Clinical Studies and Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 5Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada and Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University,

Introduction: Urothelial carcinoma (UC) is the most common urinary bladder tumor in dogs. Despite combination therapies, most patients succumb to local tumor progression or metastatic disease. The programmed death receptor (PD-1) and its ligand (PD-L1) have been studied in a variety of human cancers. Consequently, therapies directed at PD-1 and/or PD-L1 are now incorporated into the treatment of muscle invasive UC in people. Presently, limited data exists about the expression of PD-1/PD-L1 in dogs, especially in UC. Therefore, the goal of this study was to evaluate the expression of PD-1 and PD-L1 in canine UC cell lines and PD-1 on lymphocytes from peripheral blood and urine of dogs with UC.

Methods: Three canine UC cell lines were evaluated for the presence of PD-1 and PD-L1 expression via western blot, flow cytometry, and fluorescence microscopy. The presence of PD-1 on lymphocytes from peripheral blood and urine was evaluated via flow cytometry in 10 dogs with UC, 10 healthy control dogs, and 4 dogs with urinary tract infections.

Results: PD-1 and PD-L1 expression were detected in all 3 cell lines. Lymphocyte PD-1 expression was increased 2.4-fold on CD8+ T-cells in the urine of dogs with UC compared to healthy dogs (p = 0.0006). Dogs with UC had a trend towards increased T-regulatory cells in both blood and urine compared to healthy control dogs (p = 0.11).

Conclusion: Detection of PD-1 / PD-L1 in canine UC cell lines and the upregulated expression in canine clinical patients supports future work as a possible therapeutic target in UC.

Funding Information: Funding for this project was provided by the OVC Pet Trust.

64 Friday, October 16, 2020

International Oral Abstracts

65 MINIMAL DOSE EFFICACY OF PEG-L-ASPARAGINASE IN HEALTHY BEAGLE DOGS

Presenter Name: Laurien Feenstra Presenter Institution: Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands Presenter Email: [email protected]

Authors: Laurien Feenstra1, Ronette Gehring2, Inge van Geijlswijk2,3, Ada Krupa4, Berthil Prinsen5, Tim Lammens6, Erik Teske1

1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands 2 Division of Veterinary Pharmacotherapy and Pharmacy, Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands 3 Pharmacy Department, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands 4 AniCura Animal Hospital Zeeuws-Vlaanderen, Terneuzen, Netherlands 5 Department of Genetics, section Metabolic Diagnostics, UMC Utrecht, Utrecht University, Utrecht, Netherlands 6 Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium

Introduction: L-asparaginase is a frequently used drug in L-CHOP protocols in the treatment of canine malignant lymphoma. Since production and availability of native E-Coli derived L-asparaginase is limited in various European countries, PEG-L-asparaginase (PEG-ASP) is a commonly used alternative. The objective of this study is to find the minimal dosage and dose interval of PEG-ASP in dogs.

Materials and methods: Multi-phase clinical dose finding study with seven healthy Beagle dogs. Plasma amino acid concentrations (including asparagine and aspartic acid) and PEG-ASP were measured at various time points after subcutaneous administration of different dosages PEG-ASP.

Results: Administration of 10IU/kg PEG-ASP resulted in asparagine suppression in all dogs for various durations; 9 days in all seven dogs, 15 days in five dogs, 21 days in three dogs and 29 days in one dog. Subsequent administration of a second dose of 20IU/kg PEG-ASP resulted in asparagine suppression shorter than 9 days in five dogs. Plasma asparagine suppression was seen at PEG-ASP plasma concentrations of 22IU/L.

Conclusions: There appears to be great individual variation in response to PEG-ASP. A dose of 10IU/kg PEG-ASP was already sufficient in all dogs for a minimal asparagine suppression of 9 days, although a great variation existed in the maximal period of suppression. Previously thought to be less immunogenic than L-asparaginase, resistance to PEG-ASP seems to appear frequently and can occur after one injection. Although PEG-ASP plasma concentrations of 100IU/L are considered the minimal therapeutic level in humans, asparagine suppression occurs already with PEG- ASP plasma concentrations of less than 25IU/L in the canine samples.

66 CANINE TRANSMISSIBLE VENEREAL TUMOR: RETROSPECTIVE STUDY OF 50 CASES DIAGNOSED DURING THE PERIOD OF 2016- 2019 IN MEXICO

Presenter Name: Eliza Vazquez-Sanchez Presenter Institution: Universidad Nacional Autonomal de Mexico (UNAM) and Hospital de Urgencias Veterinarias Borges Presenter Email: [email protected]

Authors: Vazquez-Sanchez, Eliza1

1Universidad Nacional Autonomal de México (UNAM) and Hospital de Urgencias Veterinarias Borges

Introduction: ORAL Canine transmissible venereal tumour (CTVT) affects primarily external genitalia of dogs, but also can be transplanted to skin, subcutis, liver, spleen, nasal cavity and others tissues. It is the only naturally contagious tumour that can be transmitted to skin or mucosal areas during coitus, licking, sniffing, scratching, and biting. Its metastatic potential is low (less than 5%). CTVT first arose from somatic cells of an individual wolf or a dog that lived in east Asia more than 10,000 years ago and perpetuates as a parasitic allograft in the host.

Aim of study: The aim of this study was to determine epidemiological characteristics of CTVT in Mexico.

Methods: This is a retrospective study consisting in 576 patients diagnosed with tumours at the oncology area of the Small Animal Hospital of the National University of Mexico and Hospital de Urgencias Veterinarias Borges during the period of 2016 - 2019.

Results: From the total count of 576 patients, 50 of them presented CTVT. This number accounts for 8.6% of the oncology patients. The highest incidence was seen in these breeds: mixed breed 46% (23), Pitbull 20% (10) and Boxer 14% (7). The most frequent age where the tumor appeared was 3 and 4 years of age. Male dogs were found to be more affected than females, where 44% (22) were intact males, 18% (9) neutered males, 28% (14) intact females and 10% (5) spayed females. The majority presented genital CTVT, but was also diagnosed on the skin, nasal and oral cavity Only one patient was diagnosed with metastasis to a lymphnode similar to what has been reported in other studies.

Conclusions and clinical significance: CTVT showed a high incidence in adult and intact dogs in a reproductive stage. CTVT is frequently diagnosed in Mexico. This information will help create alternatives for the control and reduction of the CTVT prevalence.

67 PHASE I DOSE ESCALATION STUDY OF 12B80 – HYDROXYBISPHOSPHONATE LINKED DOXORUBICIN – IN DOGS WITH NATURALLY OCCURRING OSTEOSARCOMA

Presenter Name: Pierre Boyé Presenter Institution: Department of Small Animal Teaching Hospital, The Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh Presenter Email: [email protected]

Authors: Boye, Pierre1,2,3, David, Emmanuelle4, Le Bot, Ronan4 , Serres, François1,2, Marescaux, Laurent2, Tierny, Dominique1,2

1 OCR (Oncovet-Clinical-Research), Loos France 2 Oncovet, Villeneuve d’Ascq, France 3 Department of Small Animal Teaching Hospital, The Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh 4 Atlanthera, Saint Herblain, France

Introduction: 12b80 is a new antineoplastic compound, combining doxorubicin to a bone targeting hydroxybisphosphonate vector with a pH-sensitive linker, designed to specifically trigger doxorubicin release in acid bone tumor microenvironment. Preclinically, 12b80 displays in vivo stronger antitumor effects on rodent orthotopic osteosarcoma compared with doxorubicin/zoledronate combination. This phase I study was aimed to determine the safety and toxicity profiles of 12b80 in dogs with naturally occurring osteosarcoma, with the objective to translate findings from dogs to humans.

Materials and methods: Client-owned dogs with untreated osteosarcoma were evaluated in an accelerated dose titration design followed by 3+3 design, to determine the safety, tolerability, maximum tolerated dose (MTD), and dose-limiting toxicity (DLT) of 12b80 given intravenously, every three weeks for three cycles.

Results: Ten dogs were enrolled and treated at 4 mg/kg (n = 1), 6 mg/kg (n = 2), 8 mg/kg (n = 3), and 10 mg/kg (n = 4). The MTD of 12b80 was 8 mg/kg (i.e. equivalent calculated dose of doxorubicin of 110 mg/m2, range: 93 – 126). No DLT was observed at this dose level. Most adverse events included grade 1 or 2 gastrointestinal disorders and hypersensitivity reactions. No hematologic and cardiac DLT were observed at any dose level tested.

Conclusions: This study showed that 12b80 is overall well tolerated in dogs, expanding the therapeutic index of doxorubicin up to four times the standard dose of 30 mg/m2 of doxorubicin. The results show potential translational relevance for further clinical developmentof 12b80 in dog and human osteosarcoma.

68 EN BLOC RESECTION OF EXTENSIVE ADRENAL PHEOCHROMOCYTOMA INCLUDING THE CAUDAL VENA CAVA IN DOGS

Presenter Name: Kazushi Asano Presenter Institution: Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Japan Presenter e-mail: [email protected]

Authors: Kazushi Asano1, Kumiko Ishigaki1, Orie Yoshida1, Takahiro Nagumo1, Kei Tamura1

1Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Japan

Introduction: Canine pheochromocytomas (PHEO) often infiltrate into the intrahepatic caudal vena cava (CVC) at the time of diagnosis. The purpose of this study was to describe the surgical procedure and outcome of en bloc resection of extensive adrenal PHEO including the CVC in dogs.

Materials and methods: Four dogs (#1 - #4) with adrenal mass preoperatively diagnosed by computed tomography (CT) were included. All cases had an extensive adrenal mass invading into the intrahepatic CVC. In addition, CT revealed multiple collateral formation of venous returns caused by the tumor thrombus. In all cases, en bloc resection of the adrenal mass including the CVC was planned. The medical records of all cases were reviewed.

Results: The adrenal mass was the right side in cases #1, #2 and #3, and the left side in case #4. Intraoperative femoral venous pressure was continuously measured. For the en bloc resection, Pringle maneuver, preadrenal CVC occlusion, and/or intrathoracic CVC occlusion were performed in all cases. In addition, the occlusion of right renal vein was combined in case #4. En bloc resection of adrenal mass including the CVC was achieved in all cases. The operation time was between 135 and 212 minutes. Postoperative prognosis was good in all cases except for case #3 that died of acute renal failure on postoperative 4th day.

Conclusions: The feasibility of en bloc resection of extensive adrenal PHEO including the CVC might be determined based on CT findings of collateral venous return and intraoperative monitoring of femoral venous pressure.

69 MACHINE LEARNING FOR EARLY DETECTION OF HEMANGIOSARCOMA

Presenter Name: Taylor DePauw Presenter Institution: Animal Cancer Care and Research Program, University of Minnesota (USA) Presenter Email: [email protected]

Authors: DePauw, Taylor1,2,3, Khammanivong, Ali1,2,3, Modiano, Jaime F.1,2,3,4,5

1Animal Cancer Care and Research Program, University of Minnesota 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota 3Masonic Cancer Center, University of Minnesota 4Stem Cell Institute, University of Minnesota 5Center for Immunology, University of Minnesota

Introduction: We developed a test to detect circulating hemangiosarcoma-associated cells in dogs prior to disease onset. Dogs were assigned into risk categories for hemangiosarcoma development, allowing for the rational deployment of eBAT, a drug targeting cancer-stem cells and the tumor niche, as a means for cancer prophylaxis.

Methods: 125 dog samples (28 hemangiosarcoma; 29 other cancers; 27 benign vascular pathology; and 41 healthy) comprised a training set for machine learning. 10-fold cross-validation established parameters for hemangiosarcoma- associated cell detection and determined sensitivity and specificity. We applied these algorithms to a prospective cohort of 209 clinically healthy golden retrievers, boxers, and Portuguese Water Dogs as a validation set to assess test performance.

Results: Classification accuracy was 85% for healthy dogs and up to 89% for hemangiosarcoma. The test has outstanding specificity (95%) and acceptable sensitivity (89%) for early detection: the false negative rate at 6-months after testing was <1% (1/99). Twenty-one dogs in the prospective cohort developed cancer or another chronic condition; nineteen with a prediction of cancer-associated pathology were diagnosed with cancer (90%). Two of these 21 dogs (10%) were misclassified. One had a cancer prediction but died from a non-malignant condition. The other had a “healthy” prediction but died of cancer.

Clinical Significance: To our knowledge, this is the first test that accurately assigns risk for hemangiosarcoma development in clinically healthy dogs, providing rationale for cancer chemoprophylaxis. Our study also provides proof-of-concept for prospective trials for early cancer detection in companion dogs.

70 Abstract removed from this page.

71 PREDICTING LIKELIHOOD OF IN VIVO CHEMOTHERAPY RESPONSE IN CANINE LYMPHOMA USING EX VIVO DRUG SENSITIVITY AND IMMUNOPHENOTYPING DATA IN A MACHINE LEARNING MODEL

Presenter Name: Sungwon Lim Presenter Institution: ImpriMed, Inc. Presenter Email: [email protected]

Authors: Zach Bohannan1, Raghavendra Pudupakam1, Jamin Koo1,2,3, Harrison Horwitz1, Josephine Tsang1, Amanda Polley1, James Enyang Han1, Elmer Fernandez1, Stanley Park1, Deanna Swartzfager1, Nicholas Seah Xi Qi1, Chantal Tu 4, Wendi Velando Rankin4, Douglas H. Thamm5, Hye-Ryeon Lee1,2, and Sungwon Lim1,2

1ImpriMed, Inc., USA, 2ImpriMed Korea, Inc., Seoul, Republic of Korea, 3Department of Chemical Engineering, Hongik University, Republic of Korea, 4SAGE Veterinary Centers, USA, 5Flint Animal Cancer Center, USA

Introduction: For a life-threatening disease like cancer, optimal treatment may need to be personalized for each patient. We developed a precision medicine platform that evaluates the probability of chemotherapeutic efficacy for canine lymphoma by combining ex vivo chemosensitivity and immunophenotyping assays with computational modeling. This study evaluated the correlation between our ex vivo data-driven prediction and reported responses in a series of canine lymphoma patients.

Methods: We collected live cancer cells from fresh fine needle aspirates taken from affected lymph nodes and collected post-treatment clinical responses in 261 canine lymphoma patients who were scheduled to be treated with at least 1 of 5 common chemotherapy agents (doxorubicin, vincristine, cyclophosphamide, lomustine, and rabacfosadine). Samples were subject to flow cytometry analysis for immunophenotyping and ex vivo chemosensitivity testing. For each drug, 70% of the treated patients were randomly selected to train a random forest model to predict the probability of positive Veterinary Cooperative Oncology Group (VCOG) clinical response based on input variables including antigen expression profiles and treatment sensitivity readouts for each patient’s cancer cells. The remaining 30% of patients were used to test model performance.

Results: Most models showed good performance by ROC-AUC and Brier score. Predictive efficacy scores significantly distinguished (P < 0.0001) positive responses from negative responses, both when comparing B- vs. T-cell patients and naïve vs. relapse patients. In addition, the patient group with predictive efficacy scores > 50% showed a statistically significant reduction (log-rank P < 0.05) in time to complete response when compared to the group with scores < 50%.

Conclusions: The computational model developed in this study enabled conversion of ex vivo cell-based assay results into probability of in vivo therapeutic efficacy, which may help improve treatment outcomes in individual canine lymphoma patients by providing predictive estimates of positive treatment response.

72 Abstract removed from this page.

73 AN EXPLORATORY INVESTIGATION OF NOVEL MOLECULAR TARGETS FOR CHEMOTHERAPY IN CANINE HISTIOCYTIC SARCOMA

Presenter Name: Hirataka Tomiyasu Presenter Institution: Department of Veterinary Internal Medicine, the University of Tokyo (Japan) Presenter Email: [email protected]

Authors: Asada, Hajime1, Tomiyasu, Hirataka1, Watanabe, Kei2, Matsumoto, Yuki2, Irie, Mitsuhiro3, Harada, Kei4, Kobayashi, Tetsuya4

1Department of Veterinary Internal Medicine, the University of Tokyo (Japan) 2Anicom Speciality Medical Institute Inc., (Japan) 3Shikoku Veterinary Medical Center (Japan) 4Japan Small Animal Cancer Center (Japan)

Introduction: To identify novel molecular targets for chemotherapy in canine histiocytic sarcoma (HS) using next generation sequencing.

Methods: Tumor samples obtained from dogs suffered from HS were subjected to whole exome sequencing (WES) (8 samples) and RNA-sequencing (4 samples). With respect to a candidate molecule, FGFR1, its relative mRNA expression level was compared between HS tumor samples and normal monocytes. Expression of FGFR1 protein was also examined by immunohistochemistry using 13 samples. Finally, effects of FGFR1 inhibitors were examined in cultured canine HS cell lines.

Results: In WES, 39 gene mutations were identified, and pathway analysis using these genes revealed that gene mutations were significantly concentrated in PI3K-Akt and ERK1/2 pathways. Based on these observations, we investigated the expression levels of receptor tyrosine kinases (RTKs) using the data of RNA-sequencing, leading to a finding that FGFR1 mRNA expression level was higher than those of other RTKs in HS tumor tissues. In addition, FGFR1 mRNA level was significantly higher in HS tumor cells than normal monocytes. Immunohistochemistry revealed the expression of FGFR1 in tumor tissues from all of the 13 dogs examined. Finally, treatment with FGFR1 inhibitors induced cell cycle arrest, increase of apoptosis, and inhibition of proliferations in 3 canine HS cell lines.

Conclusions: The intracellular signaling from FGFR1 and mutations of genes associated with the regulations of PI3K- Akt and ERK1/2 pathways might be involved in the pathogenesis of canine HS.

Clinical significance of the results: FGFR1 might be a novel molecular target for chemotherapy in canine HS.

74 ELECTRO-IMMUNOTHERAPY IN VETERINARY MEDICINE: ACTIVATING LOCAL AND SYSTEMIC IMMUNITY

Presenter Name: Luigi Aurisicchio Presenter Institution: 2Evvivax (Italy) Presenter Email: [email protected]

Authors: Impellizeri, Joseph A.1, Salvatori, Erika2, Palombo, Fabio3, Conforti, Antonella2, Aurisicchio, Luigi2,3

1Veterinary Oncology Services (USA) 2Evvivax srl (Italy) 3Takis srl (Italy)

Introduction: Electrochemotherapy (ECT) is an approved treatment for local control of accessible tumor nodules. Recently, calcium electroporation (ECaT) has shown efficacy in clinical trials in humans and in vet species. Nonetheless, ECT/ECaT results in no impact on inaccessible, distant metastases, thus resulting in no significant effects on patients’ overall survival. ECT/ECaT can activate the Immune System. However, this issue has never been investigated in details. Based on a similar technology, we have developed a genetic vaccination platform based on the use of muscle DNA electrogenetransfer (DNA-EGT). Erb-eVax is a vaccine targeting the oncogene HER2/neu, overexpressed in breast cancer and canine osteosarcoma. Here, we hypothesize that standard ECT can induce a low, but detectable immune response against Tumor Associated Antigens (TAA) and that ECT and DNA-EGT can achieve synergic effects.

Methods: we have utilized a clinically validated device for Veterinary electroporation called Vet-ePorator, based on Cliniporator technology utilized and approved in Europe for ECT/ECaT applications and adapted to EGT. Mice challenged with a breast cancer cell line overexpressing HER2 were treated with ECT/ECaT, DNA-EGT or their combination. Dogs with metastatic breast cancer or Osteosarcoma are currently being treated with ECT and/or Erb- eVax. All studies have been approved by Internal Ethics Committee for the research.

Results: ECT and ECaT induced a detectable cell-mediated immune response against HER2/neu and transient control of tumor growth. DNA-EGT was able to delay tumor growth over time whereas the combination ECT/DNA- EGT resulted in complete tumor rejection. ECT reduced tumor masses and Erb-eVax induced cell mediated and antibody immune responses against HER2/neu in treated dogs.

Conclusions: Our findings provide new data on tumor immunobiology and most importantly improve the efficacy of ECT/ECaT.

Clinical significance of the results: novel immunotherapeutic approaches with a significant impact on patient’s survival and quality of life.

Abbreviations and drug names: Erb-eVax, Calcium, Bleomycin

75 CANINE HISTIOCYTIC SARCOMA: FROM THE GENETIC PREDISPOSITIONS AND SOMATIC ALTERATIONS TO THE DEVELOPMENT OF A GENETIC RISK TEST AND THERAPEUTIC OPTIONS

Presenter Name: Benoit Hédan Presenter Institution: Institut de Genetique et Developpement de Rennes, CNRS-UMR6290, University Rennes1 (France) Presenter Email: [email protected]

Authors: Hédan, Benoit1, Cadieu, Edouard1, Rault, Mélanie1, Devauchelle, Patrick2, Abadie, Jérôme3

1Institut de Genetique et Developpement de Rennes, CNRS-UMR6290, University Rennes1 (France) 2Centre de Cancérologie Vétérinaire, MICEN Vet 58 rue Auguste Perret (France) 3ONIRIS, -Ecole Nationale Vétérinaire, Agroalimentaire et de l’Alimentation Nantes Atlantique (France)

Introduction: Histiocytic sarcoma (HS) is a rare but devastating cancer in predisposed breeds: Bernese Mountain dogs (BMD), Rottweilers and flat coated retriever (FCR). Since there is no available efficient treatment, a better understanding of the genetic mechanisms leading to HS is needed to improve treatment of affected dogs.

Material and methods: Genotyping was performed on 455 HS cases and 408 unaffected BMDs, Rottweilers and FCRs using the available SNP chips (Illumina 22K, 170K and Affymetrix 712K, SNP arrays). The Beagle software was used to impute the Illumina SNP arrays to the Affymetrix 712K SNP array. Three loci were captured and sequenced on 16 dogs with the Sureselect Target enrichment system. RNAseq was performed on 4 HS tumors and identified mutations were validated by Sanger sequencing of 100 canine HS cases. Seven in-house cell lines were developed to explore the main altered pathways.

Results: Genome-Wide Association studies on a large cohort of HS predisposed breeds confirmed the major role of CFA11 locus in HS predisposition (Shearin et al. 2012). The multi-breed approach not only refined the CFA11 locus to only 200 kb, but also allowed to identify other predisposing loci to several hematopoietic cancers. Bioinformatic analyses on three main loci are ongoing on 16 dogs. Based on these results, we developed a genetic risk test and the follow up of > 3000 BMDs tested over the 8 years-used has already showed the benefice of this genetic risk test to improve BMDs health. In parallel, we identified somatic recurrent mutations in 53% in a specific pathway and showed a link with the disseminated HS form (pvalue=8.26x10-7 Chi-square test). We then demonstrated the sensitivity of HS cell lines to available drugs targeting this pathway.

All together, these genetic results provide a better understanding of the genetics and treatment of HS in dogs.

76 CIRCULATING TUMOR DNA AS A NOVEL BIOMARKER FOR CANINE CANCERS: HISTIOCYTIC SARCOMA, ORAL MELANOMA AND LYMPHOMA

Presenter Name: Anais Prouteau Presenter Institution: CNRS-University of Rennes 1, UMR6290, Institute of Genetics and Development of Rennes, Faculty of Medicine, SFR Biosit (France) Presenter Email: [email protected]

Authors: Prouteau, Anais1, Denis, Jérôme Alexandre2, de Fornel, Pauline3, Cadieu, Edouard1, Botherel, Nadine1, François, Romain3, Dorso, Laetitia4, Devauchelle, Patrick2, Abadie, Jérôme4, André, Catherine1, Hédan, Benoît1

1CNRS-University of Rennes 1, UMR6290, Institute of Genetics and Development of Rennes, Faculty of Medicine, SFR Biosit (France) 2Sorbonne University, INSERM UMR_S938, APHP Pitié-Salpêtrière Hospital, Endocrinology and oncology Biochemistry department (France) 3Micen Vet (France) 4Oniris, Laboniris - Department of Biology, Pathology and Food Sciences, Route de Gachet (France)

Introduction: In the recent years, circulating tumor DNA (ctDNA) analysis has become an attractive noninvasive technique to monitor human cancer patients, opening a variety of clinical applications.

Aim of this study: 1) To look for the presence of ctDNA in the plasma of dogs with histiocytic sarcoma, oral melanoma and lymphoma. 2) To assess whether ctDNA can be used to monitor minimal residual disease in dogs with high-grade lymphoma treated by chemotherapy.

Methods: We collected tumor and plasma samples from 21 dogs with histiocytic sarcoma, 10 with oral melanoma and 14 with high-grade multicentric lymphoma comprising 4 dogs treated by chemotherapy. ctDNA was identified by detecting specific recurrent somatic alterations (point mutation, copy number alteration (CNA) or chromosomal rearrangement) with digital droplets PCR (ddPCR), and PCR for antigen receptor rearrangement (PARR).

Results: For histiocytic sarcomas, the targeted mutation was detected in 9/21 tumors and ctDNA was detected in all of the 9 corresponding plasmas. Concerning oral melanoma, CNA were identified in 9/10 tumors and one dog had the corresponding CNA detectable in its plasma. About dogs with lymphoma, the PARR assay was positive for 13/14 tumors, and ctDNA was detected in the plasma of 12/13 dogs at time of diagnosis. Four dogs with lymphoma received chemotherapy and minimal residual disease (MRD) was determined before each chemotherapy injection, by targeting lymphoma-specific antigen receptor rearrangement in their plasma. The detection of MRD was concordant with the evaluation of the clinical response and ctDNA analysis appears highly sensitive to assess treatment response.

Conclusions: This study shows that ctDNA is detectable in the plasma of dogs with various cancers, especially in HS and lymphoma (100% of HS and 92% of dogs with lymphoma) and is a promising biomarker for diagnosis and clinical follow-up in veterinary oncology.

77 RECOMBINANT LIVE-ATTENUATED INFLUENZA VIRUSES CO-EXPRESSING BOVINE PAPILLOMAVIRUS 1 (BPV1) E6 AND E7 INDUCE TUMOUR REGRESSION IN EQUINE SARCOID PATIENTS

Presenter Name: Sabine Brandt Presenter Institution: Research Group Oncology, Equine Clinic, University of Veterinary Medicine, Vienna, Austria Presenter Email: [email protected]

Authors: Brandt, Sabine1, Jindra, Christoph1,2, Hainisch, Edmund K.1, Wolscheck, Markus1,2, Muster, Thomas2

1Research Group Oncology, Equine Clinic, University of Veterinary Medicine, Vienna, Austria; 2 BlueSky Immunotherapies, Vienna, Austria

Introduction: Bovine papillomaviruses types 1 and 2 (BPV1, BPV2) induce semi-malignant skin tumours termed sarcoids in horses. Sarcoids seriously compromise the health and welfare of affected individuals due to their high propensity to progress upon accidental or iatrogenic trauma, and to reoccur in a more severe, multiple form following ineffective treatment.

Materials and methods: We have developed recombinant influenza (Flu) A and B viruses harbouring a partially deleted NS1 gene (iNS1) assuring live attenuation, and shuffled BPV1 E6 and E7 antigens as immunotherapeutic vaccines.

Results: In a phase I trial involving 12 healthy horses, intradermal administration of vaccines proved safe with the only transient side effect being mild fever in four horses following injection with iNS1-FluA-BPV1-E6E7 virus. Importantly, vaccine candidates also proved biologically safe: repeated screening of secretions and faeces by RT-PCR and focus forming assay during the trial testified to the absence of virus shedding. In an ongoing patient trial involving horses bearing multiple, partly recurrent sarcoids, one lesion per horse is intralesionally injected with the influenza A virus, and then boosted with the influenza B virus. Per January 2020, treatment led to highly significant tumour regression or eradication in seven, and stable disease in two of the nine patients treated thus far. Intriguingly, treatment also induced a systemic anti-tumour response in all individuals as revealed by synchronous regression or growth arrest of non-injected lesions located at different sites of the horses’ integument.

Conclusions: To our knowledge, this is the first immunotherapeutic approach showing significant reduction of the tumour burden in equine sarcoid patients.

78 79 80 Saturday, October 17, 2020

Oral Abstracts

81 MIR-190A/NRG3/ERBB4 CASCADE AS A POTENTIAL THERAPEUTIC TARGET OF CANINE GLIOMA

Presenter Name: Shunsuke Noguchi Presenter Institution: Osaka Prefecture University Presenter Email: [email protected]

Authors: Noguchi, Shunsuke1; Inoue, Marina2; Nakamoto, Yuya3; Kamishina, Hiroaki4 1Osaka Prefecture University 2Osaka Prefecture University, Izumisano, Japan 3Neuro Vets, Kyoto, Japan 4Gifu University, Gifu, Japan

Introduction: Clinical specimens used for the expression analysis of miRNA were as follows: 9 anaplastic oligodendrogliomas, 10 meningiomas, and 8 normal brain tissues. Additionally, glioma cell lines, J3T-1 and J3T-2 were used for the gene expression analysis and in vitro experiments.

Methods: Clinical specimens used for the expression analysis of miRNA were follow: 9 anaplastic oligodendrogliomas, 10 meningiomas, 8 normal brain tissues. Additionally, glioma cell lines, J3T-1 and J3T-2 were used for the gene expression analysis and in vitro experiments.

Results: The expression level of miR-190a was significantly downregulated in glioma tissues, meningioma tissues, and cell lines compared with normal brain tissues. Extrinsic miR-190a decreased the number of viable glioma cells. The expression of NRG3 and p-Akt, (which is a putative downstream target of NRG3) were decreased by the transfection with miR-190a. Also, silencing of NRG3 exhibited similar effects to extrinsic miR-190a. Luciferase activity assay revealed that NRG3 is a direct target of miR-190a. NRG3 has been shown to be a ligand of ERBB4. In fact, treatment with Afatinib, a pan-HER family inhibitor, significantly suppressed the growth of glioma cells when compared to treatment with Gefitinib, an EGFR inhibitor, or Lapatinib, an EGFR and ERBB2 inhibitor. Concurrently, Afatinib successfully decreased the level of p-Akt in glioma cells.

Conclusion: miR-190a functions as a tumor-suppressor in canine glioma by targeting NRG3, and the NRG3/ERBB4 cascade might be a promising therapeutic target of canine glioma.

Funding Information: No funding was used for this project.

82 NON-INVASIVE DETECTION OF SPONTANEOUS TUMORS IN DOGS

Presenter Name: Gillian Dank Presenter Institution: Hebrew University Presenter Email: [email protected]

Authors: Dank, Gillian1; Polliack, Gabriel 2; Aviram, Gal 2; Gur, Assaf2 1Hebrew University 2HT Bioimaging

Introduction: Presently, diagnosis of external masses is done by either fine needle aspirate or biopsy. This is invasive and in cases with multiple masses, can be costly. The HT BioImaging System is a heat diffusion system designed to record the thermal diffusivity of tissues while they are being heated and cooled. It has been calibrated to detect possible differences between benign and malignant tissues, based on their thermal unique characteristics. The purpose of this prospective study was to validate this system in dogs with external masses.

Methods: Fifty dogs with cutaneous and subcutaneous masses were recruited. The mass ( or masses) were photographed by an optical camera, measured and documented in the medical records. The HT BioImaging System procedure was then performed to evaluate the probability for malignancy in the masses. Standard diagnostics (cytology, biopsy or both) were performed in accordance with the clinical practice guidelines. The results from the clinical pathology or biopsy reports were compared to the results from the HT Bioimaging system.

Results: Analysis of 80 lesions examined from 44 dogs was performed. 6 dogs were excluded from the study. The results were Sensitivity 82.7%, Specificity 82.9%, Positive predictive value 0.77, Negative predictive value 0.87 and Accuracy 82.85%.

Conclusion: These initial, yet promising results, demonstrate the immense potential of the HT BioImaging System in differentiating between benign and malignant lesions.

Funding Information: HT Bioimaging

83 STAT3 PATHWAY IS UPREGULATED IN CANINE T-CELL LYMPHOMA

Presenter Name: Anna Luiza Facchetti Vinhaes Assumpcao Presenter Institution: University of Wisconsin-Madison Presenter Email: [email protected]

Authors: Facchetti Vinhaes Assumpcao, Anna Luiza1; Kuehnl, Ashley1; JIa, Shuai1; Pan, Xuan1 1University of Wisconsin-Madison, Madison, Wisconsin

Introduction: The Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathways play important roles in the pathogenesis of T cell lymphoma in humans and up-regulated STAT3 expression and activity are associated with worse clinical outcome in humans. No studies have evaluated the JAK-STAT signaling pathway in T cell lymphoma of dogs. The objective of this study was to evaluate if STAT3 pathway is deregulated in T cell lymphoma in dogs. We aim to assess the expression, activation, and cellular localization of STAT3 and mitogen- activated protein kinase ERK1/2 in T cell lymphoma of dogs.

Methods: Retrospective analysis of T cell lymphoma in dogs, including patient characteristics and treatment, and immunohistochemistry. Biopsy samples of 26 client-owned dogs diagnosed with T cell lymphoma by histopathology were evaluated in this study.

Results: There was a significantly higher percentage of STAT3 (62.47%) and p-STAT3 (11.29%) immunolabelled cells in canine T cell lymphoma samples compared with canine normal lymph nodes (STAT3=46.7%; p-STAT3=5.47%), (p<0.05). In STAT3 immunolabelled cells, STAT3 has higher nuclear expression in lymphoma samples than in normal lymph nodes. We are currently investigating mitogen-activated kinase ERK1/2 activation in T cell lymphoma of dogs.

Conclusion: Compared with the normal canine lymph node, T cell lymphoma of dogs has up-regulated STAT3 pathway. Our results support future investigation of JAK inhibitors in the treatment of T cell lymphoma in dogs.

Funding Information: University of Wisconsin-Madison Companion Animal Fund

84 CHARACTERIZING CIRCULATING NUCLEOSOMES IN THE PLASMA OF DOGS WITH HEMANGIOSARCOMA

Presenter Name: Heather Wilson-Robles Presenter Institution: Texas A&M University Presenter Email: [email protected]

Authors: Wilson-Robles, Heather1; Miller, Tasha1; Jarvis, Jill1; Terrell, Jason2; Dewsbury, Nathan3; Herzog, Marrielle4; Kelly, Terry2; Hardat, Nathalie4; Turatsinze, Jean-Valery4; Michel, Gaetan4 1Texas A&M University 2Volition America 3Volition Veterinary Diagnostic Development 4Belgian Volition

Introduction: Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death which causes chromatin fragmentation and release of nucleosomes into the blood. The Nu.QTM platform exploits the different compositions of circulating nucleosomes in the blood of humans that occurs with disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using the Nu.QTM platform of assays.

Methods: A total of 50 dogs diagnosed with HSA and 65 healthy controls were recruited for this study. The HSA samples were recruited from TAMU or purchased from various biobanks. All control samples were recruited from TAMU. Graphpad Prism v.8 was used to make comparisons between HSA and controls and within the HSA cohort.

Results: Dogs with hemangiosarcoma had an approximately 10-fold increase in their plasma nucleosome concentrations compared to controls (AUC 84.5%). Nucleosome concentrations increased with the stage of the disease.

Conclusion: The Nu.QTM platform was able to reliably detect elevated nucleosome concentrations in the plasma of dogs. Furthermore, it appears that nucleosomes may be useful for differentiating cancer from healthy individuals in canines. Further testing is underway to better characterize HSA and optimize the Nu.QTM platform for canine HSA detection.

Funding Information: Salary funding for Tasha Miller was provided by the Fred and Vola Palmer Chair in Comparative Oncology held by Dr. Wilson-Robles.

85 ADVERSE EVENT PROFILE OF THE THERAPEUTIC CANINE OSTEOSARCOMA VACCINE, LIVE LISTERIA VECTOR

Presenter Name: Margaret Musser Presenter Institution: Iowa State University Presenter Email: [email protected]

Authors: Musser, Margaret1; Berger, Erika1; Tripp, Chelsea2; Clifford, Craig3; Bergman, Philip4; Johannes, Chad1 1Iowa State University, Ames, IA 2Bridge Animal Referral Center, Edmonds, WA 3Hope Veterinary Specialists, Malvern, PA 4Clinical Studies, VCA; Katonah Bedford Veterinary Center, Bedford Hills, NY; Memorial Sloan-Kettering Cancer Center, New York, NY

Introduction: Osteosarcoma is an aggressive primary mesenchymal tumor of bone in both humans and dogs. Unfortunately, local and systemic treatment strategies have resulted in limited improvements in survival times, and thus alternative approaches are needed. Immunotherapy is a possible approach as osteosarcoma is known to be highly immunogenic, spurring research into therapeutic vaccine strategies. This study evaluated the safety of the lyophilized canine osteosarcoma vaccine, live Listeria vector (COV-LLV).

Methods: Participating clinicians from 11 sites completed electronic data capture forms on dogs receiving the COV- LLV during and after vaccine administration.

Results: Forty-nine cases receiving 123 doses were included for analysis. Adverse events during COV-LLV administration were typically mild and self-limiting, most commonly nausea, lethargy, and fever (veterinary cooperative oncology group – common terminology criteria for adverse events grade 1). Four dogs (8%) cultured positive for Listeria (3 infections: an amputation site abscess, a septic stifle joint, and bacterial cystitis; and one patient whose lungs cultured positive on necropsy within 24 hours of COV-LLV administration). These cases join the previously reported Listeria-positive thoracic abscess that developed at a metastatic site in a canine following administration of COV-LLV. As concurrent treatment regimens varied, evaluation of the impact of COV-LLV on overall survival time was not possible.

Conclusion: Although side effects during administration of COV-LLV were generally mild and self-limiting, clinically significant Listeria infections are possible. In addition, as Listeria is zoonotic and patients with Listeria infections may shed the bacterium; caution for veterinarians and family members of dogs receiving attenuated therapeutic vaccinations is warranted.

Funding Information: None.

86 NOVEL APPLICATION OF SINGLE CELL NEXT-GENERATION SEQUENCING FOR DETERMINATION OF INTRATUMORAL HETEROZYGOSITY OF CANINE OSTEOSARCOMA

Presenter Name: Jordan Ayers Presenter Institution: University of Florida Small Animal Hospital Presenter Email: [email protected]

Authors: Ayers, Jordan1; Cortés-Hinojosa, Galaxia2; Riva, Alberto1; Bechtel, Sandra1; Sahay, Bikash1; Cascio, Matthew1; Lejeune, Amandine1; Shiomitsu, Keijiro1; Hernandez, Oscar1; Salute, Marc1; Milner, Rowan1 1University of Florida 2Universidad de Chile

Introduction: Osteosarcoma (OSA) is a highly aggressive neoplasia of the canine and human patient. Genome sequencing has revealed OSA has a high mutational load and appears highly heterogenous. However, while bulk genome sequencing identified heterogeneity between OSA tumors, intra-tumoral heterogeneity has not been established in OSA. By performing Single-Cell Transcriptomics (SC-T), a novel, high-resolution analysis of individual cells can be generated to reveal intra-tumoral populations. This technology has not previously been applied to the canine genome. From SC-T analysis, information on tumoral subpopulations that are otherwise masked by bulk sequencing is evident.

Methods: Established cOSA cell lines were utilized for a SC-T run and the results were validated against a genomic profile generated via Sanger sequencing. The data generated from the SCT analysis were run against the canine genome via the use of HiperGator super computers. The data was then formatted and input into Ingenuity Pathway Analysis to identify pathways up or down regulated within the clusters and into Cell Loupe to generate cellular clusters.

Results: Within the ~10,000 cells analyzed from each sample, unique cellular clusters were identified. 81 pathways of relevance were identified and of those 33 had been implicated in OSA tumorigenesis—18 which had been investigated in cOSA and human OSA (hOSA) and 15 which had only been investigated in hOSA.

Conclusion: SC-T can be successfully applied to the canine genome and to cOSA. From the data, we were able to identify the expression of new pathways implicated in cOSA and gain further insight into the heterogeneity of OSA.

87 NANOPARTICLE AND LASER ABLATION OF SOLID TUMORS IN DOGS AND CATS

Presenter Name: Lisa Parshley Presenter Institution: Olympia Veterinary Specialists - Cancer Center Presenter Email: [email protected]

Authors: Parshley, Lisa1; Tripp, Chelsea 2; Hamilton, MJ3; Miller, Lisa 4; DeTaboada, Luis4; Bradley, Abbey1; Melton, Tammy5 1Olympia Veterinary Specialists -Cancer Center 2BARC 3Mobile Oncology 4LiteCure 5Olympia Veterinary Specialist -Cancer Center

Introduction: Over the last decade nanoparticles in medicine have gone from theoretical to clinical trials. Their ability to potentially target disease and to manipulate the body at a molecular level has caused much of this excitement. Nanospectra developed nanoparticles, silicone core and a gold shells, have been shown to be non-toxic and will react with near infrared light causing thermal ablation of solid tumors. In this retrospective study we evaluate efficacy and assess safety of nanoparticles and laser induced thermal ablation of a variety of tumors in dogs and squamous cell carcinoma in cats.

Methods: 65 cases treated from 2014-2020 and from three different facilities were included in the study. Data collected included signalment, toxicities, power of light used, tumor type, and responses. Animals All animals received infusion of nanoparticles followed by light therapy 24 hours later. Light therapy (810 nm) was applied to tumors using an external light source (sapphire laser). Animals were evaluated once a week for four weeks and then monthly.

Results: Laser light treated animals developed minimal toxicities, including complete death of tumor (necrosis) or mild self-resolving thermal burns. Responses varied based on tumor type, with mast cell tumors and soft tissue sarcoma having the best response. Some of the animals received a second laser application, which provided these animals longer control of their tumors.

Conclusion: Nanoparticle laser tumor ablation therapy can be applied to solid tumors with minimal side effects while providing anticancer effects

Funding Information: None.

88 VISUALIZATION AND CHARACTERIZATION OF CANCER STEM CELLS WITH LOW PROTEASOME ACTIVITY IN A CANINE OSTEOSARCOMA CELL LINE

Presenter Name: Koangyong Sung Presenter Institution: Laboratory of Veterinary Surgery, Department of Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University Presenter Email: [email protected]

Authors: Sung, Koangyong1; Hosoya, Kenji1; Murase, Yusuke1; Deguchi, Tatsuya1; Kim, Sangho1; Sunaga, Takafumi1; Okumura, Masahiro1 1Laboratory of Veterinary Surgery, Department of Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Introduction: Existence of cancer stem-like cells (CSCs) has long been proposed as a cause of treatment failure in many types of tumors. However, studies to target CSCs have been hampered by difficulty in identifying this small subpopulation of cells. Recent research to characterize CSCs have revealed that CSCs have low proteasome activity, which could be utilized for CSCs visualization. The purpose of this study was to visualize CSCs in a canine osteosarcoma cell line.

Methods: A canine osteosarcoma cell line (HMPOS) was used for this study. ZsGreen1 and carboxyl-terminal region of ornithine decarboxylase genes were transfected into the parent cells using a lentiviral system, accumulating the fluorescence in the cells with low proteasome activity, ZsG+ cells. The CSC-like properties of ZsG+ cells were evaluated on self-renewal capacity, function of ABCG2 transporter, differentiation capacity, cell cycle, and expression level of CSC markers. After irradiation, radio-sensitivity was examined, evaluating clonogenic assay, and p53-binding protein 1 (53BP1) foci formation.

Results: The accomplishment of the transfection was confirmed with inhibition of proteasome activity, the fluorescent intensity of most cells increased in HMPOS-ZsG. The CSC-like properties of ZsG+ cells showed higher self-renewal capacity, function of ABCG2 transporter, differentiation capacity, and expression level of CSC markers compared with non-ZsG+ cells. The cell cycle analysis showed low G0/G1 and high S phase of ZsG+ cells. After irradiation, ZsG+ cells were resistant against X-ray irradiation.

Conclusion: CSCs in HMPOS cells could be successfully visualized by ZsGreen1 system.

89 SOLITARY OSSEOUS PLASMACYTOMAS IN DOGS: A RETROSPECTIVE REVIEW OF 13 CASES

Presenter Name: Abby Reising Presenter Institution: University of Missouri Presenter Email: [email protected]

Authors: Reising, Abby1; Donnelly, Lindsay1; Flesner, Brian1; Maitz, Charles1; Bryan, Jeffrey1 1University of Missouri, Columbia, MO

Introduction: To characterize solitary osseous plasmacytomas (SOP) in dogs, a rare disesase, including diagnostics and treatment outcomes.

Methods: Retrospective review was conducted of records of dogs with SOP diagnosed and treated at the University of Missouri from 2004-2019. Data collected included demographics, presenting complaint, duration of clinical signs, treatment prior to referral, tumor location, pathology, diagnostics, treatment, response, date of progression and death. Kaplan-Meier estimate was used to estimate median survival time (MST) and progression free interval (PFI).

Results: Thirteen dogs were included. Median age at presentation was 8 years (range 4-11). Vertebrae were affected in 8/13 (61.5%). Primary treatment was radiation therapy in 10/13 (76.9%), with all but one treated with a definitive, finely fractionated protocol. The median number of fractions was 20 and median total dose delivered to the planning target volume was 53Gy (range 48-54) (unavailable for 6 dogs). MST for those that completed radiation therapy was 1166 days. The MST and PFI for all dogs included in the study was 912 and 331 days, respectively. For dogs with vertebral SOPs, the MST was 42 days (range 5-1898). Two of these dogs were untreated. No dogs in this case-series developed multiple myeloma.

Conclusion: This is the first study to focus solely on this rare disease. Solitary osseous plasmacytomas in dogs can often be managed long term with surgery or radiation therapy, similar to the human disease. SOP in dogs may progress to multiple myeloma less frequently than in humans.

Funding Information: No funding was utilized for this project.

90 RNA IN-SITU HYBRIDIZATION AS A MOLECULAR DIAGNOSTIC TECHNIQUE TARGETING IBA-1 AND CD204 IN CANINE HISTIOCYTIC SARCOMA

Presenter Name: Amandine Lejeune Presenter Institution: University of Florida College of Veterinary Medicine Presenter Email: [email protected]

Authors: Engelien, Julia1; Lejeune, Amandine1; Frasca, Salvatore 1; Dark, Michael1; Hernandez, Oscar1; Shiomitsu, Keijiro1 1University of Florida College of Veterinary Medicine

Introduction: Obtaining a definitive diagnosis of canine histiocytic sarcoma (HS) can be challenging. Two proteins, IBA-1 and CD204, are immunohistochemical markers helpful in distinguishing HS from other tumor types with similar morphological features. This study was performed to validate the use of a novel RNA In Situ Hybridization (ISH) technology (RNA-ISH) allowing single-molecule RNA visualization in Formalin‐Fixed Paraffin‐Embedded (FFPE) tissues and cell pellets as a diagnostic tool for the diagnosis of canine HS.

Methods: RT-PCR, genetic sequencing, Western Blot analysis and immunohistochemistry for IBA-1 and CD204 were performed to correlate gene expression and protein expression in the DH82 canine HS cell line. Once validated in the DH82 cell line, RNA-ISH for IBA-1 and CD204 was performed in clinical FFPE HS samples to demonstrate mRNA expression of these markers. FFPE archived samples of various round cell tumors (lymphomas, mast cell tumors and plasma cell tumors) and melanomas were used as negative control for RNA-ISH against IBA-1 and CD204.

Results: RNA-ISH showed moderate to strong expression of RNA for IBA-1 and CD204 genes in DH82 cell line and FFPE canine HS samples. RNA-ISH showed little to no expression of the target genes in control samples. The minimal expression of CD204 and IBA-1 seen in these tumors was assessed to be secondary to tumoral macrophagic infiltrates.

Conclusion: RNA-ISH for IBA-1 and CD204 was highly specific and sensitive. It may be a valuable diagnostic technique in the diagnosis of canine HS.

Funding Information: The Finn Project provided financial support for this study, as well as the Florida Veterinary Scholars Program and Boehringer Ingelheim Veterinary Research Scholars Program

91 RETROSPECTIVE STUDY OF FELINE MEDIASTINAL LYMPHOMA: PRESENTATION AND RESPONSE TO CHEMOTHERAPY TREATMENT

Presenter Name: Constanza Pereira Rivera Presenter Institution: Auna Especialidades Veterinaria Presenter Email: [email protected]

Authors: Pereira Rivera, Constanza1; Cebrián Pinar, Elisa1; García de la Virgen, Miguel1; Borrego Massó, Juan Francisco1 1Auna Especialidades Veterinaria, Valencia, España.

Introduction: The objective was to describe the clinical presentation, retroviral status, response to chemotherapy and survival in a population of cats with mediastinal lymphoma.

Methods: Patient characteristics of cats with cytologicallly confirmed mediastinal lymphoma in a Spanish Referral Center between 2012-2019 were collected, including: retroviral status, staging tests, chemotherapy protocol and response following RECIST criteria assessed 42 days after chemotherapy initiation, adverse effects, median time to progression (MTP) and survival times (MST).

Results: Thirty-six cases were included. Median age was 4,6 years (0.9-13 years); 29 cats were Domestic short hair (80%), 5 Siamese (13.8%), 2 Birmans and 1 Persian; male to female ratio was 1.1-1.0. Feline leukemia virus positive (FeLV+) cats represented 89% of cases (27/32) with no feline immunodeficiency affected cats. At diagnosis 18 patients had pleural effusion, 31 respiratory clinical signs and 4 gastrointestinal signs. Protocols used included COP (cyclophosphamide, vincristine, prednisolone) (n=15). Madison-Wisconsin 25 weeks including one initial dose of asparaginase (n=18), and LAP (lomustine, asparaginase, prednisolone) (n=3). Overall response rate was 47%, 13 complete responses (36%), 4 partial responses (11%). Median time to progression was 33 days (1-665 days) with a MST of 62 days (1-665 days). Adverse events evaluated were mostly gastrointestinal, being grade I, II (82%) and grade III (18%).

Conclusion: Our population had a high prevalence of FeLV-antigenaemic cats similar to older literature, which could explain our lower overall response rates, MTP and MST when compared to more recent studies, highlighting the notable geographical differences in terms of presentation and response to therapy found in this disease.

92 IN VITRO EFFECTS OF MONOACYLGLYCEROL LIPASE INHIBITION ON PHENOTYPE IN CANINE UROTHELIAL CARCINOMA CELL LINES

Presenter Name: Jordon Inkol Presenter Institution: Ontario Veterinary College/University of Guelph Presenter Email: [email protected]

Authors: Inkol, Jordon1; Mutsaers, Anthony1; Hocker, Sam2 1Ontario Veterinary College, Guelph, Ontario, Canada 2College of Veterinary Medicine, Manhattan, Kansas

Introduction: Canine urothelial carcinoma (UC) is the most common form of canine bladder cancer. Despite a variety of treatment options, patients still succumb to local disease progression and/or metastatic spread. Monoacylglycerol lipase (MAGL) is a serine hydrolase that mediates metabolism of endogenous cannabinoids. Overexpression of MAGL in several human malignancies has been associated with more aggressive and invasive tumors as it may comprise a crucial metabolic network that supports epithelial to mesenchymal transition. Our objectives were to evaluate expression of MAGL in canine UC cell lines and assess the effects of MAGL inhibition on UC phenotype.

Methods: Three canine UC cell lines (AXA, SH, and Orig) and one non-cancer canine cell line, MDCK, were evaluated. MAGL expression was assessed via Western blotting. Inhibition of MAGL was conducted with specific chemical inhibitors and DsiRNA. The half maximal inhibitory concentration for inhibitors was established using crystal violet assay. Migration and invasion capacity were assessed using transwell assays with and without Matrigel coating.

Results: MAGL expression was detected in all three UC cell lines, while MDCK showed minimal expression. AXA and Orig demonstrated low expression while SH showed high [removed]p < 0.001). Furthermore, SH demonstrated a greater migration capacity (p < 0.0001) compared to AXA and Orig. Both chemical and DsiRNA inhibition of MAGL attenuated (p <0.01) migration and invasion capacity in SH and AXA cells.

Conclusion: Targeted inhibition of MAGL in canine UC is worthy of further investigation as a therapeutic option to potentially improve outcome of patients.

Funding Information: This study was funded by a Pet Trust Operating Grant to Dr. Sam E. Hocker

93 RADIOTHERAPY ALONE VERSUS CHEMOTHERAPY FOLLOWED BY RADIOTHERAPY FOR CATS WITH NASAL LYMPHOMA.

Presenter Name: Hiroki Yamazaki Presenter Institution: Veterinary Medical Centre, College of Life, Environmental and Advanced Sciences, Osaka Prefecture University Presenter Email: [email protected]

Authors: Yamazaki, Hiroki1; Wada, Yusuke2; Tanimoto, Nanami 2; Tanaka, Toshiyuki2; Noguchi, Shunsuke3 1Veterinary Medical Centre, College of Life, Environmental and Advanced Sciences, Osaka Prefecture University 2Osaka Prefecture University 3Laboratory of Veterinary Radiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University

Introduction: It remains unclear whether radiotherapy (RT) or chemotherapy should be preferentially used as the first-line treatment for cats with nasal lymphoma (NLSA).

Methods: We retrospectively evaluated 46 cats with NLSA, of which 32 and 14 cats received RT alone, and first-line chemotherapy followed by RT, respectively. The treatment response, metastasis incidence rate, and overall survival (OS) were compared between the two groups.

Results: There were no significant differences in the demographic characteristics between both groups. The clinical signs improved in 88% (28/32) of cats that received RT and 79% (11/14) of those that received chemotherapy and RT. The overall response rate was 94% in cats that received RT, with 13 having a complete response (CR), 17 a partial response (PR), and 2 stable disease (SD). The overall response rate was 86% in cats that received chemotherapy and RT, with 4 having a CR, 8 a PR, and 2 SD. There were no significant differences in the treatment response between the groups. OS significantly increased in the cats that received RT (median: 360 days, 58-1095) compared to the cats that received chemotherapy and RT (median: 240 days, 36-1095). The incidence rate of metastasis significantly increased in 75% (9/12), and 38% (11/29) of the cats that received chemotherapy and RT, and RT, respectively. OS significantly decreased in 25 cats with metastasis (median: 191 days, 36-484) compared to the 21 cats without metastasis (median: 482 days, 120-1,095).

Conclusion: Cats with NLSA might have more improvement in their clinical outcomes with RT rather than chemotherapy.

Funding Information: The author(s) disclose receipt of no financial support for the research.

94 REPURPOSING DRUGS IN VETERINARY CANCER TREATMENT: SODIUM DICHLOROACETATE AND OMEPRAZOLE EXHIBIT SYNERGISTIC CYTOTOXIC EFFECTS ON CANINE ORAL MELANOMA CELL LINE

Presenter Name: Gabriela Toledo Presenter Institution: School of Medicine/ University of São Paulo Presenter Email: [email protected]

Authors: Toledo, Gabriela1; Nagamine, Marcia1; Dagli, Maria Lucia1 1University of São Paulo, São Paulo, Brazil.

Introduction: Sodium Dichloroacetate (DCA) is a drug which provides a shift of the Warburg effect by stimulation of oxidative phosphorylation, reduction of tumoral growth and induction of apoptosis in different cancer types. Omeprazole (OMP) is a proton pump inhibitor which in cancer has been related to the regulation of extracellular and intracellular pH, potentially reduction of chemo-resistance, the improvement of other drug’s efficacy and induction of cell death. The aim of this study is to report a cytotoxic and synergistic effect that DCA and OMP presented on a canine oral melanoma cell line.

Methods: CBMT a canine oral melanoma cell line previously established at our laboratory was treated with different concentrations of DCA and OMP alone or in combination for 72 hours. The control group was treated with DMSO or Medium only. Cytotoxic effects were analyzed through the violet crystal method and read with the 570mn spectrophotometer. Drug combination test for synergy was calculated using the Chou method and CalcSyn software to generate Combination Index (CI), CI values less than 1 are considered synergistic effects. ANOVA one-way and post testing Dunnet’s P<0.05 was considered statistically significant.

Results: The combination treatment of DCA and OMP showed higher cytotoxicity in a dose-dependent way. Every combination of DCA and OMP presented CI <1, therefore, DCA and OMP are synergistic cytotoxic drugs in CBMT cell line.

Conclusion: The combination treatment of DCA and OMP on CBMT cells exhibited cytotoxicity and synergistic effects and may be promising drugs on the treatment of canine oral melanoma.

Funding Information: This project is funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

95 COMBINING ZOLEDRONATE WITH RADIATION THERAPY TO ENHANCE THE TREATMENT OF OSTEOINVASIVE FELINE ORAL SQUAMOUS CELL CARCINOMA

Presenter Name: Alycen Lundberg Presenter Institution: University of Illinois at Urbana-Champaign Presenter Email: [email protected]

Authors: Lundberg, Alycen1; Tran Hoang, Christine1; Billhymer, Audrey1; Selting, Kimberly1 1University of Illinois at Urbana-Champaign

Introduction: Feline oral squamous cell carcinoma (FOSCC) is aggressively osteoinvasive and can be painful, with <10% one-year survival despite multi-modal therapies. Zoledronate (ZOL) is a potent bisphosphonate which reduces osteolysis and may have multiple mechanisms of radiosensitization. We hypothesized that combining ZOL with radiation therapy (RT) would be effective and well tolerated in pet cats with FOSCC and would decrease lysis.

Methods: To determine optimal timing, three FOSCC cell lines were treated with RT and ZOL at various time intervals. Cytotoxicity and clonogenicity were assessed using a sulforhodamine B assay and clonogenic assay, respectively. Pet cats with osteoinvasive OSCC were then treated with RT (8 Gy once weekly x 4, total 32 Gy) and ZOL (0.2 mg/kg IV q4wk x 2). Volumetric and longest dimension tumor response was assessed using computed tomography (CT) scans at days 0 and 50. Safety and tolerability were monitored with hematologic and biochemical assessment, and acute radiation adverse effects were characterized using VRTOG criteria. Serum CTx, a marker of bone-resorption, was measured weekly.

Results: In-vitro data revealed no clear effect on timing, therefore ZOL was given immediately prior to RT as is customary with traditional radiosensitizing agents. All 9 patients received the full treatment protocol. Evaluable responses included 1 CR, 3 PR, and 2 SD. Treatment was tolerated well in all cats with minimal RT toxicity and no ZOL toxicity. Serum CTx decreased over time in all patients.

Conclusion: Combining RT with ZOL in cats with FOSCC is safe, well-tolerated, and reduces bone resorption.

Funding Information: This work was funded by the Companion Animal Memorial Fund from the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign.

96 RETROSPECTIVE EVALUATION OF FELINE INTRANASAL CARCINOMAS TREATED WITH EXTERNAL-BEAM RADIOTHERAPY: 43 CASES

Presenter Name: Hiroto Yoshikawa Presenter Institution: NC State University Presenter Email: [email protected]

Authors: Yoshikawa, Hiroto1; Gieger, Tracy1; Saba, Corey2; Fredrickson, Kirsha2; Kubicek, Lyndsay3; Haney, Siobhan4; Ruslander, David5; Kelsey, Krista6; McEntee, Margaret7; Nolan, Michael1 1North Carolina State University 2University of Georgia 3Angell Animal Medical Center 4Hope Veterinary Specialists 5Veterinary Specialty Hospital of the Carolinas 6Carolina Veterinary Specialists 7Cornell University

Introduction: External-beam-radiotherapy (RT) is commonly used to treat feline intranasal carcinomas (FINC); however, little is known regarding the comparative efficacy of various irradiation strategies. This multi-institutional retrospective study was performed to report outcomes and identify prognostic factors associated with survival for cats with FINC.

Methods: Medical records for patients with FINC that underwent RT at one of 7 veterinary RT facilities were retrospectively reviewed. Irradiation protocols were categorized as: definitive-intent full-course RT (FRT), definitive- intent stereotactic RT (SRT), and palliative-intent RT (PRT). Median overall survival time (mOST) and disease progression-free survival (mPFS; documented via CT/MRI) were calculated. The impacts that tumor stage, RT protocol/intent, and use of adjunctive therapies had on outcome were evaluated.

Results: Total 43 cats were included (18 SRT, 8 FRT, and 17 PRT). The mOST and mPFS were 453 days (95%CI; 226-679 days) and 357 days (144-570 days), respectively. In multivariable modeling, definitive-intent treatment (FRT or SRT) was associated with significantly longer mPFS (504 days vs. 159 days for PRT; p=0.048) and mOST (721 days vs. 282 days for PRT; p=0.047). Disease laterality also associated with mPFS; unilateral tumors that were treated with a definitive-intent RT had significantly longer mPFS (542 days) than their bilateral counterpart (357 days) and uni- or bi-lateral tumors that were treated palliatively (198 and 82 days, respectively).

Conclusion: Definitive-intent RT is associated with prolonged OST and PFS as compared to PRT in cats with FINC; local tumor control is longest in cats with unilateral tumors treated with definitive-intent RT.

97 KINETICS OF CARDIAC TROPONIN I RELEASE AND CORRELATION WITH ECHOCARDIOGRAPHIC AND ELECTROCARDIOGRAPHIC PARAMETERS IN DOGS TREATED WITH SINGLE AGENT DOXORUBICIN

Presenter Name: Kim Selting Presenter Institution: University of Illinois Presenter Email: [email protected]

Authors: Wallace, Gabrielle1; Vitt, Jordan1; Fries, Ryan1; Wittenburg, Luke2; Reinhart, Jennifer1; Selting, Kim1 1University of Illinois, Champaign-Urbana, IL 2University of California, Davis, CA

Introduction: Cardiotoxicity limits the use of doxorubicin chemotherapy in dogs, often occurring after treatment is complete. No test can predict which dogs might, or will, develop cardiomyopathy. Cardiac troponin I (cTnI) increases in people after cardiotoxic chemotherapy, and peak concentrations occur within hours of treatment, subsequently returning to baseline; peak concentrations correlate with cardiac outcome. We hypothesized that cTnI would peak within 6 hours of doxorubicin administration in dogs, and that changes would be seen in global longitudinal myocardial strain. Our goal was to identify the time point at which possible predictors of cardiotoxicity should be studied.

Methods: This prospective, single-arm trial enrolled dogs greater than 10 kg scheduled to receive single-agent doxorubicin at every two-week intervals. End-of-infusion serum concentration (Cmax) of doxorubicin and doxorubicinol were assessed using high performance liquid chromatography--tandem mass spectrometry at each treatment to correlate cardiac findings with pharmacokinetics. cTnI (ultrasensitive assay), electrocardiography, and echocardiographic measures of myocardial strain and systolic function, were collected at 0, 2, 4, and 6 hours after each infusion. Cardiac function was monitored monthly for 4 months after doxorubicin completion.

Results: Dogs enrolled received 5 (n=7), 4 (n=1), or 1 (n=2) dose(s) of doxorubicin. Diagnoses included lymphoma (n=8) and solid tumors (hemangiosarcoma, n=2). Intra- and interpatient doxorubicin Cmax varied with no consistent pattern. cTnI and cardiac analyses are in progress. Doxorubicin Cmax, doxorubicinol, and echo values will be correlated with cTnI concentrations. Though not anticipated given the small number of dogs enrolled, two dogs did develop cardiac complications possibly attributable to doxorubicin.

Conclusion: Pending

Funding Information: This clinical study was funded by a resident grant from the American College of Veterinary Internal Medicine

98 Poster Abstracts

99 ADMINISTRATION REGIMEN AND FEEDING PROGRAM EFFECTS ON PHARMACOKINETIC AND PHARMACODYNAMIC PROFILES OF AN ORAL SULFORAPHANE SOURCE IN BEAGLE DOGS

Presenter Name: Nicolette Harris Presenter Institution: Nutramax Laboratories Veterinary Sciences, Inc. Presenter Email: [email protected]

Authors: Gillette, Robert1; Warner, Carolyn1; Stunk, Rebekah1; Harris, Nicolette1 1Nutramax Laboratories Veterinary Sciences, Inc. Lancaster, SC

Introduction: Vegetable consumption has been investigated in people and dogs for effects on the risk of developing Transitional Cell Carcinoma. This study examined effects of administration regimen and feeding program on pharmacokinetic (PK) and pharmacodynamic (PD) profiles of an oral proprietary tablet (Avmaquin™, Nutramax Laboratories Veterinary Sciences) containing glucoraphanin (GFN), a precursor to sulforaphane (SFN) found in cruciferous vegetables.

Methods: A lab population of beagle dogs (n=6) was used in this three-period study with a washout period between treatments and no crossover. No treatments were administered during the washout. In Phase 1 dogs orally received a dose with food, Phase 2 dogs orally received a dose BID with food, and Phase 3 dogs received a dose with food re- introduced 2 hours after administration.

Results: Data from blood samples collected at timed intervals after administration for PK analysis revealed increased plasma SFN levels in all groups, without a significant difference for Tmax (p = 0.166) or area under the curve (p = 0.097). Phase 3 had a significantly higher Cmax compared to other groups. RNA from blood samples were processed for NQO1, HO-1, GCLC, and GCLM gene expression analysis. The PD data showed that the dose increased gene expression of NQ01 and HQ1.

Conclusion: Results did not support significant difference between once and twice daily administration. Administration of GFN in a fasted state significantly increases SFN plasma levels compared to administration with food. This study supports the bioavailability of SFN source in dogs, with further studies needed to assess potential benefits for clinical applications.

Funding Information: This study was sponsored by Nutramax Laboratories Veterinary Sciences, Inc. 946 Quality Drive, Lancaster, SC, USA 29720

100 CLINICAL OUTCOME IN DOGS WITH APOCRINE GLAND ADENOCARCINOMA OF THE ANAL SAC TREATED WITH DIFFERING RADIOTHERAPY PROTOCOLS

Presenter Name: Glenna Mauldin Presenter Institution: PetCure Oncology Presenter Email: [email protected]

Authors: Mauldin, Glenna1; Mauldin, Guy1 1PetCure Oncology

Introduction: Consensus is not established regarding optimal therapy for canine apocrine gland adenocarcinoma of the anal sac (AGAAS). The purpose of this study was to better define local treatment options by comparing outcome in dogs with AGAAS treated with differing radiotherapy (RT) protocols.

Methods: Dogs with AGAAS undergoing radiotherapy at a PetCure hospital between 2015 and 2020 were studied retrospectively. Clinical data were abstracted from the medical records; adjuvant therapies were noted. Outcome was assessed using medical records and electronic follow up. Treatment related toxicities were classified according to VRTOG criteria. NCSS v. 12.0.4 was used for statistical analyses.

Results: Fifty-seven dogs were included. There were 4 RT treatment groups: hypofractionated (HF), stereotactic (SRT), conventional with surgical resection (CFRT), and fiducial guided SRT with surgical resection (SRT/Sx). The median RT-related survival time (start of RT to rescue, death or last contact) was longest in dogs that had surgery followed by CFRT or SRT (P=0.017). Statistically equivalent numbers of dogs in all groups received various chemotherapy drugs, but systemic therapy did not result in improved outcome. The most common side effects were VRTOG grades 1 and 2 acute radiodermatitis and proctitis. Six dogs had severe late term RT side effects including rectal stricture, resulting in euthanasia in 3 cases.

Conclusion: This study suggests that dogs with AGAAS that undergo surgery and CFRT or SRT have superior outcomes. Prospective studies are needed to develop techniques to minimize late term RT side effects in the rectum. Chemotherapy did not provide a definitive benefit in this study.

101 USE OF MODIFIED MOHS PASTE IN TWO CASES OF MULTICENTRIC SQUAMOUS CELL CARCINOMA IN SITU ASSOCIATED WITH PAPILLOMAVIRUS INFECTION

Presenter Name: Yasuhiro Fukuyama Presenter Institution: Azabu University Presenter Email: [email protected]

Authors: Fukuyama, Yasuhiro1; Nishiyama, Yuta1; Kawarai, Shinpei1; Fukushima, Ushio2; Ansai, Shinichi3; Madarame, Hiroo1; Shirota, Kinji 1; Maruo, Takuya1 1Azabu University 2Kamakurayama Animal Hospital, Kamakura, Japan 3Nippon Medical School Musashi Kosugi Hospital, Kawasaki, Japan

Introduction: Multicentric squamous cell carcinoma in situ (MSCCIS), known as Bowen-like disease or multiple Bowenoid in situ carcinoma, is a rare skin disease. Its pathogenesis is generally associated with papillomavirus infection. The papillomavirus shows skin tropism and persistent infection in a basal cell layer. Hence, large or multiple spread lesions are difficult to be cured by surgical excision. Zinc chloride paste (Mohs paste) is a topical fixative used for palliative treatment for accessible skin tumors. We reported for the first time two successful cases of modified Mohs paste treatment (mMPT) for a dog and cat with MSCCIS.

Methods: In Case1, a 12-year-old, spayed female domestic cat showed erosions, ulcers, and bleedings around paw pads of all feet and ventral antebrachial region of the left paw. mMPT had been repeated over once a weekly to monthly without anesthesia. In Case2, a 7-year-old, spayed female Norfolk terrier showed multiple pigmented macules and nodules at the back of the neck skin for 9 months. mMTP was repeated once per week.

Results: In Case1, the ventral antebrachial lesions were cured and progression around the paw pads was prevented by continuous mMPT. In Case2, the nodules became cutaneous ulceration, whereas the open wound was closed by epithelialization. The survival times were 514 days (Case1) and 1043 days (Case2) after the first mMPT. The side effect included mild pain during uniform application of the paste.

Conclusion: In both cases, mMPT was useful for prevention of MSCCIS progression and increasing the patients’ quality of life.

102 CHARACTERIZATION OF TUMOR ASSOCIATED MACROPHAGES IN CANINE LYMPHOMAS

Presenter Name: Sergio Vázquez Presenter Institution: Animal Health Department, University of Leon Presenter Email: [email protected]

Authors: Vázquez, Sergio1; Vallejo, Raquel1; Espinosa, José1; Callejo, Andrea1; Pérez, Valentín1 1Animal Health Department, University of Leon, León, Spain

Introduction: In human lymphomas, it has been shown that the macrophages present in the tumor have an influence on their behavior; however, studies on this topic are scarce in veterinary medicine. The aim of this work was to determine the relationship between the macrophage infiltrates with the histological grade and the immunophenotype in clinical cases of canine lymphoma.

Methods: Samples from lymph nodes of dogs with lymphoma (n = 25) were stained with hematoxylin and eosin to establish the histological grade. Immunohistochemistry was used to determine the tumor immunophenotype (CD3 and CD20 antibodies) and macrophage characterization (Iba1, CD163, iNOS and MAC387 antibodies). Macrophage counting was performed in ten high power fields per sample. Generalized linear models with Poisson distribution were used for statistical analysis.

Results: High grade and B lymphomas showed a higher number of macrophages than their counterparts. A significantly (p<0.001) higher number of macrophages (Iba1+) was detected in high grade (255.86±104.80) and B cell lymphomas (233.18±87.46). The highest numbers of M1 (iNOS+) and M2 (CD163+) macrophages were observed in B cell lymphomas. High grade lymphomas showed a higher (p<0.001) number of CD163+ cells (182.86±79.79 vs. 113.00±98.88) and recently recruited MAC387+ macrophages, that were the most abundant in T (107.15±49.55) than in B (104.67±38.26) lymphomas.

Conclusion: The number and type of macrophages present in canine lymphoma is related to the immunophenotype and the grade. In those with high grade, macrophages are actively recruited and show a predominant M2 phenotype, that has been associated with a protumoral activity.

Funding Information: none

103 OUTCOMES OF DOGS WITH LOCALIZED BILE DUCT CARCINOMA FOLLOWING SURGERY: A RETROSPECTIVE STUDY OF SEVEN DOGS

Presenter Name: Atsushi Maeda Presenter Institution: Gifu University Presenter Email: [email protected]

Authors: Maeda, Atsushi1; Goto, Sho1; Iwasaki, Ryota1; Murakami, Mami1; Sakai, Hiroki1; Mori, Takashi1 1Gifu University, Gifu, Gifu, Japan

Introduction: Anecdotally, the prognosis of dogs with bile duct carcinoma is believed to be poor, although few studies have been conducted to evaluate the outcomes of dogs with bile duct carcinoma following surgery because many cases involve metastases at initial presentation. The objective of this study was to describe the prognosis of dogs with localized massive or nodular bile duct carcinoma following surgery.

Methods: Medical records of seven dogs with massive or nodular bile duct carcinoma that underwent surgery between March 2011 and April 2019 were reviewed. None of the dogs had metastasis at first presentation. A Kaplan–Meier survival curve was generated for all dogs.

Results: Surgery was performed by partial lobectomy and complete lobectomy in six dogs and one dog, respectively. All tumors were histopathologically confirmed as completely resected. Four dogs underwent surgery alone, and three dogs were treated with surgery and adjuvant chemotherapy. Local recurrence or distant metastasis was detected in one dog. The median overall survival time was 894 days (range: 77–1386 days) with a one-year survival rate of 86% (95% confidence interval: 33–98%). The median progression-free survival time was 446 days.

Conclusion: Our results suggest that surgery provides good prognosis for massive or nodular bile duct carcinoma if the tumors are localized in the liver. Based on the present study, further investigation is needed to determine the prognostic factors.

Funding Information: There are no funders to report for this submission

104 RESPONSE AND OUTCOME OF 15 DOGS TREATED WITH TOCERANIB PHOSPHATE WITH THE RECURRENT NASAL CARCINOMA AFTER RADIATION THERAPY

Presenter Name: Yusuke Wada Presenter Institution: Osaka Prefecture University, Veterinary Medical Center Presenter Email: [email protected]

Authors: Wada, Yusuke1; Yamazaki, Hiroki1; Noguchi, Shunsuke1 1Osaka Prefecture University Veterinary Medical Center, Izumisano-shi, Osaka, Japan

Introduction: The first-line therapy for dogs with nasal carcinoma has been radiation therapy. Unfortunately, even with the treatment, most dogs with nasal carcinomas experience local progression. However, few studies have evaluated the efficacy of other treatment options for recurrent nasal carcinoma. The aim of this study is to evaluate the efficacy of the treatment with toceranib phosphate in dogs with recurrent nasal carcinoma after radiation therapy. Especially, the survival time and tumor volume were focused on.

Methods: Fifteen dogs with nasal carcinoma treated with radiation therapy were included in this retrospective study. All dogs were administered toceranib phosphate after the recurrence. Thirteen dogs were underwent CT examination to confirm recurrence,although two dogs were without CT examination.

Results: Radiation treatments was delivered in a daily protocol of 10×4.4 Gy for 10 dogs, and weekly protocol of 4×8.0 Gy for five dogs. Toceranib phosphate was administered 3 times a week at a dose of 2.3-3.1 mg / kg. Eleven of 13 dogs examined with CT examination after administration of toceranib phosphate experienced clinical benefit (1 complete response, 5 partial response, 5 stable disease), and the clinical signs of all of 2 dogs without CT examination were improved. Overall median survival time (MST) was 506 days. MST for dogs with stage IV was 506 days. Median progression free interval (PFI) after administration of toceranib phosphate was 139 days.

Conclusion: This study indicated that toceranib phosphate improved the clinical signs of the dogs with nasal carcinoma and MST of the dogs with stage IV nasal carcinoma that recurred after radiation therapy, compared with outcomes of them treated with radiation therapy alone in some previous studies.

105 ANTI-CANCER EFFECT OF FENBENDAZOLE BY INDUCING G2/M ARREST AND MITOTIC SLIPPAGE ON CANINE MELANOMA CELL LINES

Presenter Name: Se-hoon Kim Presenter Institution: Chungnam National University Presenter Email: [email protected]

Authors: Kim, Se-hoon1; Seo, Kyoung-won1 1Chungnam National University

Introduction: Oral melanoma is one of the most common malignant tumors in the oral cavity in dogs. Fenbendazole is one of the benzimidazole drugs and is used as a safe anthelmintic drug to treat various parasites. Anti-parasitic effects of fenbendazole result from differences in the structure of tubulin in parasites and mammalian cells, and higher affinity of fenbendazole to tubulin in parasites. Meanwhile, the anti-cancer effects of fenbendazole in mammalian cells were recently reported. Fenbendazole disrupts tubulin and mitotic spindle formation in rapidly dividing tumor cells which results in apoptosis. This study aims to evaluate in vitro anti-cancer effect of fenbendazole in five canine oral melanoma cell lines.

Methods: Five canine melanoma cell lines originated from oral cavity were used. Cell viability was evaluated by MTS assay at 48 hours of fenbendazole treatment. Cell cycle was analyzed by flow cytometry with propidium iodide/RNase solution during 24 hours of treatment. Western blot analysis was used to find an apoptotic effect with cleaved Poly ADP ribose polymerase (PARP).

Results: All melanoma cell lines exhibited reduced cell viability in a dose-dependent manner. A significant decrease in the number of cancer cells was detected at 1 μM fenbendazole treatment and viability decreased below 20% at 100 μM treatment. Cell cycle analysis of all cell lines showed G2/M arrest and increased sub-G1 population. Western blot analysis exhibited that apoptosis was evident from cleaved PARP.

Conclusion: Fenbendazole treatment induces cell cycle arrest in the G2/M phase and decreases cell viability via apoptosis.

Funding Information: This research was carried out with the support of “Cooperative Research Program of Center for Companion Animal Research (Project No. PJ014045022020): Rural Development Administration, Republic of Korea.

106 INTRAPERITONEAL ADMINISTRATION OF SYNTHETIC MICRORNA-214 EXHIBITS TUMOR SUPPRESSION IN AN INTRAPERITONEAL DISSEMINATION MOUSE MODEL OF CANINE HEMANGIOSARCOMA

Presenter Name: Yoshikawa Ryutaro Presenter Institution: Laboratory of Veterinary Clinical Oncology, Joint Department of Veterinary Medicine, Gifu University Presenter Email: [email protected]

Authors: Ryutaro, Yoshikawa1; Atsushi, Maeda1; Yoshihito, Ueno1; Hiroki, Sakai1; Tomohiro, Sawadaishi2; Satoru, Kohgo2; Kohei, Yamada2; Takashi, Mori1 1Gifu University, Gifu, Gifu, Japan. 2Yamasa Corporation, Choshi, Chiba, Japan.

Introduction: MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate various biological processes. We previously developed a clinically applicable synthetic microRNA-214 (miR-214/5AE) for canine hemangiosarcoma (HSA). In this preliminary study, we evaluated the effect of intraperitoneal injections of miR-214/5AE in an intraperitoneal dissemination model of HSA.

Methods: The HSA cell line, Re21, was used for in vivo assessment. miR-214/5AE was developed using the methods previously described. Re21 cells were inoculated intraperitoneally. Two weeks after the cells were inoculated, miR- 214/5AE or nonspecific miRNA (2 nmol) in 150µL OptiMEM was mixed with 10 µl of Lipofectamine RNAiMAX, and the mixture was injected intraperitoneally every 2 days for a total of four times. The control group received injection of nonspecific miRNA, and the treatment group received injection of miR-214/5AE (each group contained two mice). The mice were then sacrificed, and peritoneal dissemination was evaluated by counting the number of tumors and the tissues were processed for histological examination.

Results: In the treatment group, the number of disseminated foci was lower than that in the control group. (control: 152, 103; treat: 31, 72) Furthermore, the number of Ki-67 positive cells was lower in the treatment group than in the control group. (control: 19, 19%; treatment: 6, 12%) The protein expressions of caspase-3 and p53 in the treatment group were higher than those in the control group.

Conclusion: These findings indicated miR-214/5AE induces tumor growth suppressive effects and apoptosis in vivo.

107 RAPAMYCIN ANTI-TUMORAL EFFECT ON PRIMARY AND METASTATIC CANINE MAMMARY GLAND TUMOR CELLS IN VITRO

Presenter Name: Carlos Fonseca-Alves Presenter Institution: Paulista University - UNIP Presenter Email: [email protected]

Authors: Lainetti, Patricia1; Leis-Filho, Antonio1; Kobayashi, Priscila1; Laufer-Amorim, Renee1; Souza, Fabiana1; Fonseca-Alves, Carlos 2 1Sao Paulo State University - UNESP, Botucatu, Sao Paulo, Brazil 2Paulista University - UNIP

Introduction: Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due the rapamycin antitumor potential, the aim of this study was to evaluate the effect of rapamycin on preclinical model of primary mammary gland tumors from female dogs and their respective metastases.

Methods: Two samples from primary tumors and two paired metastases were cultured in vitro, characterized as tumor cell lines and treated with rapamycin. Cells were evaluated for AKT, mTOR, PTEN and 4EBP1 gene [removed]qPCR) and investigated for rapamycin IC50.Then, the four cell lines were treated with rapamycin IC50 dosage and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and proteomic analysis by mass spectrometry.

Results: MTT assay demonstrated rapamycin IC50 for the different tumor cells between 2 and 12 M. In the cell lines treated with rapamycin IC50, there was an AKT increased expression after treatment, suggesting a cellular positive feedback to its blockade, which may indicate a mechanism of activation of alternative pathways. The proteomic analysis of cells treated with rapamycin revealed 32 differentially expressed proteins (p <0.05) between the groups (treated versus untreated cells). The three most relevant proteins were phosphoglycerate mutase and L-lactate dehydrogenase that were decreased in the group treated with rapamycin, both are related to cellular metabolic processes, and Myotrophin that was found increased in the same group, which is associated with positive regulation of cell growth.

Conclusion: The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor cells in vitro, however, concentrations needed to reach the IC50 were high, when compared to other studies. Thus, the in vivo rapamycin dosage necessary to inhibit tumor cells should exceed the maximum recommended dosage in vivo.

Funding Information: This research was founded by São Paulo State Research Foundation (#2015/25400-7)

108 CELL CYCLE GENES AS POTENTIAL PROGNOSTIC BIOMARKERS IN CANINE MAMMARY CANCER

Presenter Name: Barbara Borges Presenter Institution: Universidade Federal do Pará Presenter Email: [email protected]

Authors: Pinheiro, Danilo1; Sousa, Raissa2; Pereira, Washington Luis3; Aguirra, Lucien Roberta3; Harada, Maria Lucia2; Burbano, Rommel Mario2; Borges, Barbara4 1UFRA, Paragominas, Para, Brasil 2UFPA, Belem, Para, Brasil 3UFRA, Belem, Para, Brasil 4Universidade Federal do Pará

Introduction: Mammary cancer is the most common cancer of intact female dogs. Due to its high incidence and malignancy rate, characterization of biomarkers for clinical use is a priority in veterinary medicine. The aim of this work was to assess the gene expression patterns of eight genes (CCNA2, CCNB2, CHEK2, TTK, TP53, P21, MDM2 and TGFb-1) in canine mammary cancer and their relationships with clinical parameters to verify their feasibility as prognostic markers.

Methods: Eighty-four samples (44 tumoral and 40 non-tumoral) from 40 individuals plus two animals with no history of cancer were studied. Molecular subtypes (Luminal-A and -B, HER-2+ and Triple Negative) were classified by immunohistochemistry. The quantification of gene expression was performed using real-time PCR. Correlations between the results and clinical parameters were assessed statistically, with significance set at p ≤0.05. All the procedures were approved by the local ethics committee on animal use (CEUA-23084.000265/2013-53-UFRA).

Results: Differences in gene expression were observed in all the studied genes except TGFb-1. No correlations between gene expression and clinical characteristics were observed except for MDM2 and pseudopregnancy and P21 and 1-year survival. Correlations between molecular subtypes and gene expression levels were observed in CCNA2 and MDM2 (Luminal-types) and TTK (Triple Negative), while CCNB2 and CHEK2 high expression were observed regardless the molecular subtype. In humans, high expression levels of TTK, CHEK2 and CCNA2 are associated with docetaxel, trastuzumab and tamoxifen resistance, respectively.

Conclusion: Our results suggest these genes could be used as prognostic markers, especially when choosing appropriate chemotherapy protocols.

Funding Information: CNPq, CAPES, UFPA, UFRA

109 IDENTIFICATION OF SENTINEL LYMPH NODES USING CT LYMPHOGRAPHY IN DOGS WITH ORAL MALIGNANT MELANOMA

Presenter Name: Ryota Iwasaki Presenter Institution: Gifu University Presenter Email: [email protected]

Authors: Iwasaki, Ryota1; Goto, Sho1; Mori, Takashi1 1Gifu University, Gifu, Japan

Introduction: In canine oral malignant melanoma (OMM), it is essential to accurately identify the sentinel lymph nodes (SLNs) that receive drainage from the primary tumor at diagnosis. However, the lymphatic pathways in the head are complex, with multiple lymphocentra receiving drainage. We hypothesized that SLNs can be detected using CT lymphography (CTLG).

Methods: Dogs with OMM that had undergone CTLG, excluding cases with distant metastasis, were enrolled. CTLG was performed using the following procedure: first, CT scans, including the primary tumor and the regional lymph nodes, were obtained; second, according to tumor size, approximately 1−2 ml of a 300 mgI/ml iodinated contrast agent was injected around the tumor; finally, CT scans were performed 0, 3, 6, and 9 minutes after injection.

Results: Out of 60 dogs, 59 had their SLNs successfully identified. Thirty-one dogs had a single SLN, and 28 dogs had more than two SLNs. Although the ipsilateral mandibular lymph node (iMLN) was always detected in each dog, the remaining SLN locations in the dogs that had more than two SLNs were inconsistent with the primary tumor location or size. On the univariate analyses, the tumor size and the palate-invading tumor were identified as significant variables associated with the number of SLNs (P = 0.003 and < 0.001, respectively).

Conclusion: Almost all SLNs were detected by CTLG. Although iMLN should be sampled in all dogs to confirm whether metastasis is present, the possibility that multiple SLNs exist needs to be considered when a larger or palate-invading tumor is discovered.

110 EFFECT OF STIMULATOR OF INTERFERON GENES (STING) SIGNALING IN CANINE AND HUMAN OSTEOSARCOMA ON CYTOKINE/CHEMOKINE EXPRESSION AFTER RADIATION EXPOSURE

Presenter Name: Sita Withers Presenter Institution: Louisiana State University Presenter Email: [email protected]

Authors: Withers, Sita 1; Moeller, Cambri1; Liu, Chin-Chi1; Quick, Cally 1; Baham, Shelby1; Subramanian, Ramesh1; Merkle, Jennifer 1; Looper, Jayme1; Kousoulas, Konstantin1 1Louisiana State University, Baton Rouge, LA

Introduction: In response to detection of cytosolic double-stranded DNA STING signaling induces type I interferon and chemokine expression, and promotes T lymphocyte recruitment. STING is therefore critical for inducing an immune response to cells experiencing DNA damage. The objectives of this study were to determine the integrity of STING signaling in canine and human osteosarcoma (OSA) cell lines, and to correlate STING expression with cytokine/chemokine expression after radiation.

Methods: We utilized four human OSA cell lines (U2OS, MG63, SAOS-2, SAOS-2_LM6), three canine OSA cell lines (D17, Khepri, Brutus), and human and canine osteoblasts. STING expression was quantified by Western blot and mass spectrometry, and pathway integrity was tested by exposing cells to 10 μg/ml of 2’3’-cGAMP and measuring downstream transcripts by RT-qPCR, and chemokine excretion by ELISA. Cells were exposed to 0, 5, or 10 Gy and expression of downstream cytokine/chemokines was quantified by RT-qPCR at multiple timepoints.

Results: STING is frequently downregulated in human and canine OSA cell lines. STING-deficient cell lines display dampened expression of CCL5 and CXCL10 after treatment with the STING agonist 2’3’-cGAMP. CCL5 upregulation in irradiated cells is consistently dampened in the STING-deficient SAOS-2 cell line, while robust increases are seen in the STING-proficient SAOS-2_LM6 cell line at day 3 and 6. Additional data collection is in progress.

Conclusion: The integrity of STING signaling in OSA cells may affect the degree of immune modulation induced by radiation. Further studies are necessary to determine if STING signaling in OSA cells influences the anti-tumor immune response within the complex tumor microenvironment.

Funding Information: Research reported in this abstract was supported by the LSU School of Veterinary Medicine Competitive Research Program.

111 PILOT STUDY TO EVALUATE EFFICAY AND TOXICITY FOR AN ADJUVANT DOSE- INTENSIFIED DOXORUBICIN PROTOCOL IN DOGS WITH HEMANGIO SARCOMA FOLLOWING SPLENECTOMY

Presenter Name: Miguel Garcia de la Virgen Presenter Institution: Aúna Especialidades Veterinarias Presenter Email: [email protected]

Authors: Garcia de la Virgen, Miguel1; Borrego Massó, Juan Francisco1 1Aúna Especialidades Veterinarias, Paterna, Valencia, Spain

Introduction: The purpose of this prospective study was to evaluate the toxicity and efficacy of a dose- intensified adjuvant doxorubicin protocol in canine splenic hemangiosarcoma.

Methods: Inclusion criteria required a histopathological diagnosis, complete staging (stage I, II and III without extraabdominal metastasis), physical examination (PE), chest x-rays (CXR), abdominal ultrasound (AUS), echocardiography and complete bloodwork. Data collected included clinical variables at each visit, drug doses, adverse events (AE) (VCOG-CTCAE), median progression free survival (PFS) and overall survival (OS). Doxorubicin was administered at 30mg/m2 or 1mg/kg (<15kg), every 10 days, for five total administrations, with maropitant at 2mg/kg/day PO for four days. Echocardiography, AUS and CXR were repeated every two administrations and every four months thereafter.

Results: Ten dogs were included, stage I (n=1), stage II (n=8), stage III (n=1). Six males and 4 females, average weight was 20.49kg and average age of 11.1 years. Toxicity was mild with 13/17 grade I/II, and four grade III/ IV AE, (gastrointestinal (n=8), hematological (n=8), cardiac (n=1)). One grade IV gastrointestinal AE resulted in protocol discontinuation. One of the three dogs living more than a year developed transient ventricular tachycardia grade I responsive to atenolol. Two dogs received the maximum planned dose intensity 3.6mg/m2/ day, seven had modifications because of weight (n=4) or AE (n=3), receiving 2.6mg/m2/day and 3.1-3.4mg/m2/ day respectively. The median PFS and OS were 173 and 275 days respectively.

Conclusion: A dose-intensified every 10 days doxorubicin protocol was well tolerated with similar PFS and OS times reported previously although a larger population is necessary to evaluate its efficacy.

112 OWNER ATTITUDES TOWARD SCREENING FOR EARLY CANCER DETECTION IN PET DOGS

Presenter Name: Jason Chibuk Presenter Institution: PetDx Presenter Email: [email protected]

Authors: Chibuk, Jason1; Holtvoigt, Lauren1; Grosu, Daniel1; Polk, Arthur1; Chorny, Ilya1; Flory, Andi1 1PetDx

Introduction: Cancer is the leading cause of canine death, affecting up to 1 in 3 dogs. Early cancer detection can save lives by allowing for surgical removal of localized malignancies. Non-invasive pan-cancer screening tests are being developed for humans and may soon be available for dogs.

Methods: Two independent dog owner surveys were conducted by GroupSolver (n=2,004) and Frost & Sullivan (n=506). Similar open- and closed-ended questions in each survey focused on cancer awareness, willingness to treat, and trusted information sources.

Results: Survey results were comparable, with 61-73% correctly perceiving canine cancer as a common problem. Most owners see value in early cancer screening, and the majority (53-60%) would pursue some form of intervention with an early cancer diagnosis, including surgery and/or chemotherapy. Owners are generally willing to treat early stage cancer, with an average willingness to spend of $3,455, including 18% willing to spend over $5,000. Willingness to pursue cancer screening in their canine companions was more highly associated with pet humanization and bonding than educational or income level. Both surveys conclusively identified the veterinarian as the most trusted information source (78-83%) followed by family/friends, pet stores and websites.

Conclusion: Most dog owners are aware of the high risk of cancer in dogs, and the value of early detection for achieving better outcomes. Importantly, the most trusted source of information about health and wellness screening is the veterinarian. Novel pan-cancer screening tests, when available, are likely to be adopted by a significant proportion of dog owners.

Funding Information: This study was funded by PetDx.

113 TOXICITY EVALUATION OF ALTERNATE-DAY TREATMENT WITH TEYSUNO (TEGAFUR/ GIMERACIL/OTERACIL) IN DOG WITH SOLID TUMOR

Presenter Name: Takuya Maruo Presenter Institution: Azabu University Presenter Email: [email protected]

Authors: Maruo, Takuya1; Fukuyama, Yasuhiro1; Nishiyama, Yuta1; Nemoto, Yuki1; Yoda, Shinichiro1; Hosaka, Soshi1; Kayanuma, Hideki1; Orito, Kensuke1 1Azabu University

Introduction: The fluoropyrimidine 5-fluorouracil (5-FU) is infrequently used to manage tumors in veterinary oncology. Teysuno (TS-1) is an oral fixed-dose combination of three active substances – tegafur, gimeracil, and oteracil. After absorption, tegafur is metabolized into 5-FU. This combination is intended to minimize toxicity. The purpose of this study was to evaluate the safety of Teysuno in dogs with solid tumors.

Methods: A regimen of 2.5 mg/kg BID was administered to each dog with solid tumors on alternating days (Monday-Wednesday-Friday).

Results: Nine dogs were included. The initial dose of TS-1 (mean ± SD) was 2.3 ± 0.5 mg/kg administered BID three days (M-W-F) per week. The median administration period was 67 days (range, 14 – 491 days). Cause of discontinuation was death (n = 3), decreased QOL due to adverse events (n = 3), and tumor progression (n = 3). Tumor response was noted PR (n = 1), SD (n = 1), PD (n = 5), and not assessed (n = 2). Other beneficial effects were noted in one dog: seizures were controlled for 49 days. Adverse events were noted in 5 dogs, including scleral pigmentation (n = 4), anorexia related to olfactory loss (n = 2), skin pigmentation (n = 2), diarrhea (n = 1), increased T-Bil (n = 1), and opacified cornea (n = 1).

Conclusion: Teysuno was safe in tumor bearing dogs, and adverse events were mild. Efficacy was detected in some cases. Therefore, it may be a viable option for treating malignant solid tumors in dogs.

Funding Information: No funding was used for this project.

114 RE-IRRADIATION AFTER HYPOFRACTIONATED RADIOTHERAPY FOR RECURRENT INTRANASAL TUMORS IN DOGS: A RETROSPECTIVE STUDY

Presenter Name: Yuta Nishiyama Presenter Institution: Azabu University Presenter Email: [email protected]

Authors: Nishiyama, Yuta1; Maruo, Takuya1; Nemoto, Yuki1; Fukuyama, Yasuhiro1; Ueno, Hirona1; Hosaka, Soshi1; Kayanuma, Hideki1 1Azabu University

Introduction: Most dogs with intranasal neoplasia show a favorable tumor response to radiotherapy (RT); however, the long-term prognosis is poor. The progression-free interval (PFI) of hypofractionated RT is shorter than that of fractionated RT. Re-irradiation may be indicated after hypofractionated RT. The purpose of this study was to investigate re-irradiation after hypofractionated RT for recurrent intranasal tumors.

Methods: This was a retrospective study on re-irradiation after hypofractionated RT in dogs with intranasal tumors.

Results: Eighteen dogs met the selection criteria. Tumor types were adenocarcinoma (n = 13), transitional cell carcinoma (n = 2), papillary adenocarcinoma (n = 2), chondrosarcoma (n = 1). Tumor stages were I (n = 1), II (n = 5), III (n = 8), and IV (n = 4). The median number of fractions and dose/fraction of the first course of RT were 4 and 7.5 Gy, respectively. The median number of re-irradiation courses and dose/fraction were 2 and 7.5 Gy, respectively. The median total isocenter dose, total number of fractions, PFI, and overall survival were 58 Gy, 9, 383 days (range, 84–1,433 days), and 1,600 days (range, 262–1,606 days), respectively. The early side effect of re-radiation was alopecia (grade 1), and late side effects were skin problems (grade 1) and vision changes (grades 1 and 2). Dogs with a PFI exceeding 1 year survived longer than dogs with a PFI of less than 1 year (P = 0.0291).

Conclusion: Re-irradiation after hypofractionated RT was effective in dogs with recurrent intranasal tumors.

115 INTRATUMORAL TREATMENT OF CANINE HIGH-GRADE MAST CELL TUMORS WITH TIGILANOL TIGLATE (EBC-46).

Presenter Name: Graham Brown Presenter Institution: QBiotics Group Limited Presenter Email: [email protected]

Authors: Brown, Graham1; Campbell, Justine1; Jones, Pamela1; Johannes, Chad2; Reddell, Paul1 1Qbiotics Group Limited, Yungaburra, Queensland, Australia 2Iowa State University, Ames, Iowa

Introduction: To assess the efficacy of tigilanol tiglate as a local treatment for canine high-grade mast cell tumors (HGMCT).

Methods: Eighteen dogs with single tumors cytologically diagnosed as HGMCT were treated with tigilanol tiglate (1 mg/mL in buffered 40% propylene glycol). Dosing was based on tumor volume (0.5 mg tigilanol tiglate/cm3 tumor volume). The drug was delivered intratumorally using a Luer lock syringe and a fanning technique to maximise distribution throughout the tumor mass. Efficacy was assessed on the presence/absence of a complete response (full tumor resolution) at days 28 and 84 using Response Evaluation Criteria in Solid Tumors (RECIST). For dogs not achieving a complete response after 28 days, the protocol was repeated with a second intratumoral tigilanol tiglate injection.

Results: Nine out of 18 dogs (50%) in the study achieved and maintained a complete response to at least Day 84 after their first or second treatment. Five of these dogs received a single treatment, four required a second injection. Adverse events were low grade, transient and largely associated with drug’s mode of action.

Conclusion: Tigilanol tiglate showed efficacy for local treatment of HGMCT, complete response rate was improved with a second injection. HGMCT are often associated with a poor prognosis. Tigilanol tiglate provides an alternative treatment approach particularly for patients where lesion location dictates that surgical margins are not achievable and surgical candidates with an unacceptable anaesthetic risk.

Funding Information: QBiotics Group Limited provided all funding.

116 THE EFFECT OF ARGINASE ON CANINE LYMPHOCYTE FUNCTION AND ITS MODULATION BY VINCRISTINE CHEMOTHERAPY IN DOGS WITH LYMPHOMA

Presenter Name: Aimee Soileau Presenter Institution: Louisiana State University Presenter Email: [email protected]

Authors: Soileau, Aimee1; Costa, Victoria1; Moeller, Cambri1; Boudreaux, Bonnie1; Withers, Sita1 1Louisiana State University, Baton Rouge, Louisiana

Introduction: Arginase expression by myeloid-derived suppressor cells (MDSCs) is known to inhibit T lymphocyte functions in humans and mice with cancer. The objectives of this study were to determine the effect of arginase on canine lymphocyte cytotoxicity and proliferation, and to evaluate the effect of vincristine chemotherapy on blood MDSC prevalence and arginase expression in dogs with lymphoma.

Methods: Peripheral blood mononuclear cells (PBMCs) isolated from cancer-bearing dogs were exposed to increasing concentrations of arginase (0-100 nM) in the presence or absence of concanavalin A (5 ug/ml). After 2 days, flow cytometry was performed to evaluate changes in CD3, CD4, CD8, CD45RA, CD62L, granzyme B, CD25, and Ki67 expression on lymphocytes. Additionally, PBMCs were collected from dogs with naïve, multicentric, high-grade lymphoma prior to, and one week post, vincristine chemotherapy (without concurrent prednisone). Response to vincristine was prospectively assessed. The proportion of MDSCs (CD11b+CD14-MHCII-) and MDSC expression of arginase was measured by flow cytometry.

Results: Arginase decreased expression of granzyme B on CD8+ T lymphocytes, and inhibited CD4+ and CD8+ T lymphocyte proliferation. Replicates of these experiments are pending. Vincristine chemotherapy induced a decrease in the percentage of MDSCs in 3 of 4 dogs and decreased arginase expression in MDSCs of all 4 dogs. Analysis of additional samples and correlation with response is still underway.

Conclusion: These preliminary results suggest vincristine chemotherapy, or reduction in lymphoma burden, may lead to a decreased prevalence of circulating MDSCs and reduction of their arginase expression, which may alleviate T lymphocyte suppression in these dogs.

Funding Information: Research reported in this abstract was supported by the LSU Department of Veterinary Clinical Sciences Competitive Research Program.

117 TREATMENT OF INDOLENT CUTANEOUS T CELL LYMPHOMA (CUTANEOUS LYMPHOCYTOSIS) WITH HYPOFRACTIONATED RADIATION THERAPY IN THREE CATS

Presenter Name: Laura Chadsey Presenter Institution: The Ohio State University Presenter Email: [email protected]

Authors: Chadsey, Laura1; Cook, Matthew1; Green, Eric1; Jennings, Ryan 1; Moore, Peter2; Vernau, William2; Kisseberth, William1; Diaz, Sandra1 1The Ohio State University, Columbus, OH 2University of California, Davis, CA

Introduction: Feline indolent cutaneous T cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. The objective is to report the clinical outcome of three cats with ICL treated with hypofractionated radiotherapy (RT).

Methods: Medical records and client surveys were reviewed. ICL was diagnosed based on histopathology and further confirmed using immunohistochemistry and PCR for antigen receptor rearrangement (PARR). All cats were treated with hypofractionated RT (4 fractions of 8 Gy).

Results: All cats presented with skin lesions characterized by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Prior to hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T cell receptor gamma gene rearrangement. After RT, two cats had histologic improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histologic resolution of lesions with only minimal residual lymphocytes. One cat was determined to have complete clinical response with resolution of erythema and hair re-growth. Two cats had partial clinical responses; of which, one cat developed progressive disease 202 days after RT, with skin lesions including pruritus and ulceration. No cats had clinically relevant adverse events secondary to RT. All clients reported improvement in their cat’s quality of life.

Conclusion: Clinical and histologic improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

118 CLINICAL OUTCOME AND PATHOLOGY OF CANINE THYROID CARCINOSARCOMA (2006-2020)

Presenter Name: Molly Gasparini Presenter Institution: Colorado State University Presenter Email: [email protected]

Authors: Cook, Matthew1; Gasparini, Molly2; Cianciolo, Rachel1; Brown, Megan1; Moore, Anthony3; Curran, Kaitlin 4; Maxwell, Elizabeth5; Griffin, Lynn2; Gasson, Shelby6; Jansenss, Brandan 6; Veluvolu, Sridhar7; Keepman, Samuel8; Wouda, Raelene9; Selmic, Laura1 1The Ohio State University, Columbus, OH 2Colorado State University, Fort Collins, CO 3Veterinary Oncology Consultants, Lake Innes, New South Wales, Australia 4Oregon State University, Corvallis, Oregon 5University of Florida College of Veterinary Medicine, Gainesville, FL 6Texas A&M University, College Station, Texas 7University of California - Davis, Davis, CA 8University of Wisconsin School of Veterinary Medicine, Madison, WI 9Kansas State College of Veterinary Medicine, Manhattan, KS

Introduction: Carcinosarcomas are malignant mixed tumors that contain both epithelial and mesenchymal cells components on histopathology. These tumors have been reported in the mammary gland, lung, salivary gland, uterus and, in at least 8 case reports, the thyroid of dogs.

Methods: In this retrospective study, we describe the clinical presentation, treatment, and outcome of 14 cases of canine thyroid carcinosarcoma (TCS). Histopathologic samples were collected from 10/14 cases and reviewed by a single board-certified anatomic pathologist (histopathological data collection in progress.)

Results: All 14 dogs had curative-intent surgery to remove the thyroid neoplasm. The most common surgery performed was marginal excision with a unilateral thyroidectomy (10/14 dogs). Post-operatively, systemic therapy was administered in 8 dogs. Six dogs developed local recurrence of the TCS, with a median time to recurrence of 156 days (95% CI: 49-not calculated). Ten dogs developed metastatic disease with the most common metastatic site being the lungs (6/9 dogs), with a median time to metastasis of 92.5 days (0-not calculated days). Ten dogs were euthanized due to local or distant progression of the TCS. The overall median survival time was 156 days (95% CI: 49-244). The median survival time for dogs treated with adjuvant therapy was 189 days (95% CI: 24-244) while dogs without adjuvant therapy had a median survival time of 156 days (95% CI: 35-156; p=0.62).

Conclusion: TCS are an uncommon neoplasm of the dog and appear to have a poor prognosis after surgical excision with or without adjunctive therapy.

Funding Information: No funding was used for this project.

119 EVALUATION OF THE SAFETY AND EFFICACY OF CORIOLUS VERSICOLOR POLYSACCHAROPEPTIDE (I’M YUNITY®) ALONE OR IN COMBINATION WITH DOXORUBICIN FOR DOGS WITH SPLENIC HEMANGIOSARCOMA AND ITS ABILITY TO PROLONG SURVIVAL

Presenter Name: Allison Gedney Presenter Institution: University of Pennsylvania Presenter Email: [email protected]

Authors: Gedney, Allison1; Krick, Erika2; Martins, Rene3; Scavello, Heather1; Salah, Pascale1 1University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 2Mount Laurel Animal Hospital, Mount Laurel, NJ 3Penn Medicine, Center for Cellular Immunotherapies, Philadelphia, PA

Introduction: Splenic hemangiosarcoma (HSA) is an aggressive, highly metastatic tumor of vascular endothelium which carries a grave prognosis with surgery and standard of care (SOC) Doxorubicin (DOX). This is an expansion of a previous phase I study, which showed evidence of prolonged survival. We hypothesized that IMY alone and in combination with DOX may prolong survival.

Methods: This was a prospective, randomized double blind, placebo and SOC controlled trial. Dogs were eligible if they underwent splenectomy with a life expectancy ≥4 weeks. Owners opted for IMY alone or SOC DOX therapy (open label). Dogs receiving DOX were then randomized to the addition of IMY or placebo (blinded). All were monitored weekly for 15 weeks, then monthly until death.

Results: 101 dogs were included in the final analysis, 25 DOX, 51 IMY, and 25 in the IMY with DOX groups. An interaction between treatment group and sex revealed that for males, there was no significant difference in Hazard Ratios (HR) amongst groups. Females receiving DOX alone had significantly lower HR compared to those receiving IMY alone (HR 0.21; p=0.004). Dogs with HCT ≤ 30 (HR5.28; p<0.001) at screening and stage III dogs (HR 2.9; p=0.014) had significantly higher HRs when controlling for sex and treatment group. Overall, IMY was well tolerated alone and in combination with Doxorubicin.

Conclusion: When controlling for HCT and stage, female dogs treated with IMY alone had reduced survivals compared to DOX treatment groups; in males there was no significant difference identified between SOC vs IMY based treatments.

Funding Information: This study was funded by the makers of I’m Yunity®.

120 3D CELL CULTURE – ON TOP METHOD AS A RELIABLE PRECISION THERAPY MODEL IN CANINE MAMMARY TUMORS

Presenter Name: Debora Aparecida Pires de Campos Zuccari Presenter Institution: Faculty of Medicine of Sao Jose do Rio Preto, Sao Jose do Rio Preto, Sao Paulo, Brazil Presenter Email: [email protected]

Authors: Godoy, Bianca1; Colombo, Jucimara1; Moschetta-Pinheiro, Marina1; Novais, Adriana1; Zuccari, Debora1 1Faculty of Medicine of Sao Jose do Rio Preto, Sao Jose do Rio Preto, Sao Paulo, Brazil

Introduction: Canine mammary tumors (CMT) are frequently diagnosed and share many similar features with human breast cancer, thus serving as an excellent clinical model for potential precision treatment’s discovery. PI3K/ AKT/mTOR intracellular signaling is a relevant cell cycle regulatory pathway, but mutations are commonly observed in CMT. We searched a model to evaluate the effectiveness of certain chemotherapeutic treatments using 3D cell culture, due to the cells’ ability to organize themselves in a three-dimensional way, allowing the pharmacological performance of the medicine to be more authentic.

Methods: Primary cell culture was established by trypsin enzymatic disaggregation of tumor fragments from a female dog Grade 2 Mixed Tumor Carcinoma. Cells were cultured in Matrigel using 3D on top method. Formed organoids were submitted to a 72 hour interval treatments using 5 and 10 µM concentration of Erlotinib, Cetuximab and Carboplatin (gold standard).

Results: Both 5 and 10 µM Erlotinib treated organoids showed significant morphological changes. After 72 hours, 10 µM Erlotinib treated organoids showed complete degradation and death, proving effectiveness of this drug. No visible changes were observed in the other treatments.

Conclusion: The EGFR inhibitors Cetuximab or Erlotinib were used as a precision treatment for PI3K/AKT/mTOR pathway mutations. Our results suggest Erlotinib was more effective than the other treatments. Therefore, it proved to be a feasible and easily reproduced model on a case-by-case basis, directed towards precision treatment.

Funding Information: São Paulo Research Foundation (FAPESP)

121 DESIGN, ANALYTICAL VALIDATION, AND DIAGNOSTIC YIELD OF A NOVEL CANINE CANCER GENE SEQUENCING PANEL

Presenter Name: William Hendricks Presenter Institution: Translational Genomics Research Institute (TGen) Presenter Email: [email protected]

Authors: Wong, Shukmei1; Warrier, Manisha1; Byron, Sara1; Zismann, Victoria1; Boateng, Martin1; Facista, Salvatore1; Whitsett, Timothy1; Tallant, Colt1; Briones, Natalia1; Banovich, Kathryn1; Haworth, David1; Chuang, Han-Yu1; Hendricks, William1 1Translational Genomics Research Institute (TGen)

Introduction: Canine cancer genome sequencing studies are unearthing new candidate diagnostic, prognostic, and predictive biomarkers. Development and rigorous validation of genomic diagnostics is thus increasingly important for enabling new biomarkers to be confidently leveraged in research and the clinic.

Methods: We have designed a next-generation sequencing, hybrid-capture, canine gene panel covering 482,000 base pairs of 120 genes associated with canine or human cancer. We are performing analytical validation via reproducibility (inter-batch, -instrument, and -operator), sensitivity (limit-of-detection and limit-of-blank), specificity (interfering substances, probe specificity, cross-contamination), and concordance (cross-platform and -tissue, positive/negative percent agreement) testing. We are also constructing and validating a cloud-based bioinformatic pipeline utilizing truthset and cross-platform genomic data for confident calling of single nucleotide variants, copy number variants, and internal tandem duplications in tumor cells from fine needle aspirates and formalin fixed paraffin embedded tissue. We are determining diagnostic yield in >200 tumor, tissue, and breed types. Candidate somatic, pathogenic mutations are annotated with diagnostic, prognostic, and predictive associations based on a “knowledgebase” comprising 1,680 biomarker associations curated through natural language and manual processing of 272 canine publications in addition to inference via translated protein alignments and conservation scoring from the Catalogue of Somatic Mutations in Cancer (COSMIC) and from curation of 394 human publications (in the Clinical Interpretation of Variants in Cancer database, CIViC).

Results: Data to be presented.

Conclusion: We have developed a sensitive and specific gene sequencing panel capable of detecting candidate biomarkers in a wide range of canine cancer and tissue types.

Funding Information: These studies have been funded by Vidium Animal Health, a subsidiary of the Translational Genomics Research Institute (TGen).

122 CLINICAL BIOMARKERS OF CANCER CACHEXIA IN CATS

Presenter Name: Caitlin Jozwiak Presenter Institution: University of Pennsylvania School of Veterinary Medicine Presenter Email: [email protected]

Authors: Church, Molly1; Jozwiak, Caitlin2; Michel, Kathryn3; Krick, Erika4 1University of Pennsylvania School of Veterinary Medicine Department of Pathobiology, Philadelphia, PA, USA 2University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 3University of Pennsylvania School of Veterinary Medicine Department of Clinical Sciences and Advanced Medicine, Philadelphia, PA 4University of Pennsylvania School of Veterinary Medicine Department of Clinical Studies, Philadelphia, PA

Introduction: Cancer cachexia (CC) is a multifactorial syndrome that occurs in human cancer patients, and is associated with involuntary weight loss, anorexia, sarcopenia, and increased serum levels of inflammatory cytokines and acute phase proteins (APP). No effective interventional treatments currently exist. Some cats with cancer present with clinical signs similar to humans with CC; however, specific clinical biomarkers in cachectic cats have not been investigated.

Methods: In this study, we aim to characterize abnormalities indicative of CC in cats by comparing body weight, body condition, complete blood count, serum chemistry, and inflammatory cytokine concentrations in cachectic and non-cachectic cats with cancer, and healthy cats. Additionally, we aim to evaluate clinical follow up data to determine prognostic value of measured biomarkers.

Results: Initial analysis reveals mean body weight and BCS were not significantly different, demonstrating the importance of measuring muscle mass in clinical evaluation of CC. Cachectic cats had significantly lower hematocrit and albumin compared to non-cachectic (p<0.0001, p<0.0001) and healthy cats (p<0.0001, p=0.0003). Cancer- bearing cats had significantly higher white blood cell counts compared to healthy cats (p<0.0001). Cachectic cancer- bearing cats had significantly higher serum amyloid A compared to both non-cachectic and healthy cats (p=0.0086, p=0.05). Analysis of alpha1-acid glycoprotein is in progress; we hypothesize cachectic cancer-bearing cats will have elevated alpha1-acid glycoprotein, and cats with elevated APP will have shorter overall survival.

Conclusion: Results thus far indicate the clinical and inflammatory profiles of feline CC is similar to humans, signifying potential for a feline model to further research of CC in human cancer patients.

Funding Information: Mary Lowe Center for Comparative Oncology Seed Grant University of Pennsylvania, School of Veterinary Medicine

123 124 CLINICAL CHARACTERISTICS AND OUTCOMES OF LYMPHOMA WITH MOTT CELL DIFFERENTIATION IN MINIATURE DACHSHUNDS

Presenter Name: Aki Ohmi Presenter Institution: The University of Tokyo Presenter Email: [email protected]

Authors: Ohmi, Aki1; Tanaka, Miho1; Rinno, Jun2; Tsuboi, Masaya1; Chambers, James3; Uchida, Kazuyuki3; Goto- Koshino, Yuko4; Tomiyasu, Hirotaka4; Ohno, Koichi4; Tsujimoto, Hajime4 1Veterinary Medical Center, The University of Tokyo, Tokyo, Japan 2Nikotama Animal Hospital, Tokyo, Japan 3Laboratory of Veterinary Pathology, The University of Tokyo, Tokyo, Japan 4Department of Veterinary Internal Medicine, The University of Tokyo, Tokyo, Japan

Introduction: Lymphoma with Mott cell differentiation (MCL) is known to be an uncommon variant of canine lymphoma. Although it has been described in several case reports/series, there is no comprehensive study to characterize this type. In Japan, we sometimes experience cases with MCL in Miniature Dachshunds (MDs), a popular breed in our country. This study was aimed to demonstrate its clinical characteristics and outcomes.

Methods: Medical records of MDs diagnosed with lymphoma at Veterinary Medical Center of the University of Tokyo (2008 – 2017) were evaluated. MCL was diagnosed when cytology revealed neoplastic cells in lymphoma to differentiate into Mott cells having abundant Russel bodies in cytoplasm.

Results: Medical records of 113 MDs diagnosed with lymphoma were collected. Cytology specimens could be re- evaluated in 87 dogs. Of the 87 dogs, 9 dogs (10%) were diagnosed with MCL. All 9 dogs were categorized into alimentary lymphoma from the tumor locations (small and/or large intestine, 6 dogs; mesenteric lymph node, 3 dogs). Median age was 3 years old (range, 1 – 9 years). All 9 dogs were treated with chemotherapies including (L-)CHOP, melphalan and chlorambucil. Overall response rate was 78% (7/9). Overall survival in these 9 dogs ranged from 6 to >1,513 days (median: 240 days), significantly longer that that in 29 MDs with alimentary large cell lymphoma other than MCL (range: 0 – >3,801 days, median: 57 days) (P=0.0491).

Conclusion: Alimentary MCL was occasionally diagnosed in relatively young MDs. Its prognosis was better than that of alimentary lymphoma other than MCL.

Funding Information: No funding was used for this project.

125 SAFETY AND TOXICITY OF SMALL MOLECULE INHIBITORS IN DOGS WITH SPONTANEOUSLY OCCURRING MALIGNANCIES—THE USE OF REAL WORLD DATA

Presenter Name: Gerald Post Presenter Institution: FidoCure Presenter Email: [email protected]

Authors: Harvey, Garrett1; Lambert, Lindsay1; Rodrigues, Lucas1; Lewis, Ben1; Lopes, Christina 1; Post, Gerald1 1FidoCure, Palo Alto, CA

Introduction: The FidoCure ® platform integrates canine tumor genomics with access to small molecule, targeted therapeutics. The use of small molecule inhibitors (SMIs), given their success in treating cancers in people, is an attractive therapeutic opportunity in canine cancer. The purpose of this investigation was to evaluate the adverse event profile of 8 SMIs (trametinib, rapamycin, lapatinib, dasatinib, imatinib, vorinostat, toceranib and crizotinib) in tumor- bearing canine patients enrolled through FidoCure ®.

Methods: Proposed initial doses for these SMIs were gleaned from the veterinary literature (ie. imatinib, rapamycin, dasatinib, vorinostat and toceranib) or from the respective drug’s FDA and/or EMA approval package’s NOAEL established for normal dogs. Adverse events (AEs) were collected from patient clinical records and graded mild, moderate, or severe based upon clinical characteristics including degree of therapeutic intervention required for resolution, changes to quality of life, and adjustment to dose or interval of the targeted therapy.

Results: Of 464 patients with clinician reported outcomes, 156 patients (34%) reported a suspected adverse event. Across all agents, AEs were predominantly mild (61%) with only 4 AEs graded as severe. Many of these pets were treated with a combination of cytotoxic chemotherapy and SMIs. The most commonly reported adverse events include lethargy, anorexia, vomiting, diarrhea, anemia, and/or liver enzyme elevation.

Conclusion: The adverse event profiles of the SMIs enabled by FidoCure are comparable to previously published reports of the safety of Palladia ® used alone or in combination with cytotoxic chemotherapy. Studies categorizing results according to the VCOG-CTCAE criteria and research evaluating the PK and PD of these drugs are underway.

126 CERTIFICATION OF HERITABILITY OF FAMILIAL GASTROINTESTINAL POLYPOSIS IN JACK RUSSELL TERRIER AND ITS CLINICAL SIGNS.

Presenter Name: Wakana Yoneji Presenter Institution: Nara Animal Referral Clinic Presenter Email: [email protected]

Authors: Yoneji, Wakana1; Hirata, Akihiro2; Yoshizaki, Kyoko 2; Yoneji, Kensuke1; Nisii, Naohito3; Sakai, Hiroki2; Nishida, Shuhei1 1Nara Animal Referral Clinic 2Laboratory of Veterinary Pathology,Joint Department of Veterinary Medicine,Faculty of Applied Biological Sciences,Gifu University 3Laboratory of Veterinary Internal Medicine,Joint Department of Veterinary Medicine,Faculty of Applied Biological Sciences,Gifu University

Introduction: This is a report showing that a disease similar to human Familial Adenomatous Polyposis Coli exists in a particular dog breed, the Jack Russell Terrier, and proves this disease is a familial inherited disease. In dogs affected, single to many polyps can be found from the stomach to the large intestines. In addition, it has been proven that an adenocarcinoma sequence occurs in the polyps over time.

Methods: Eight related Jack Russell terriers were evaluated in this study. All cases received the genetic test. The gene mutation that causes this disease is known to be an APC gene mutation in exon 4. The mutation is autosomal dominant.

Results: Six of eight JRTs were positive for the genetic mutation and the average age of positive for the test was 5.3 years. The symptoms of affected dogs included bloody stool, rectal excretion and vomiting occurring a while after eating. Affected dogs had hematological changes including low albumin, anemia, and elevated white blood cell count. Four of six JRTs positive for the mutation were found to have many adenocarcinoma lesions in the GI simultaneously. These dogs tended to live longer after diagnosed than typical gastric adenocarcinoma cases. NSAID’s are effective for this disease in the short term, but long-term use leads to gastric bleeding.

Conclusion: We believe that this disease exists not only in Japan but also in the world based on the analysis of patient’s pedigrees. A good understanding of this disease is important to reduce its incidence in this breed.

127 THE INTEGRATED CANINE DATA COMMONS: AN OPEN PUBLIC DATA SHARING PLATFORM

Presenter Name: Matthew Beyers Presenter Institution: Frederick National Laboratory for Cancer Research Presenter Email: [email protected]

Authors: Beyers, Matthew1; Musk, Philip1; Kim, Erika2; Jensen, Mark1; LeBlanc, Amy2; Mukherjee, Amit1; Otridge, John1; Knapp, Deborah3; Parchment, Ralph1; Singh, Anju2; Sommers, Connie2; Hecht, Toby2 1Frederick National Laboratory for Cancer Research 2National Cancer Institute 3Purdue University College of Veterinary Medicine

Introduction: Novel targeted therapies and immunotherapies are emerging that hold considerable promise for cancer patient treatments. Highly relevant pre-clinical animal models are essential to identify, refine and inform the most promising candidates to advance into human clinical trials. In many cases, canine cancer closely mimics the human condition in pathology, molecular features, biological behavior, host immunocompetence, and treatment response.

Methods: The NCI’s Division of Cancer Treatment and Diagnosis (DCTD) has launched the Integrated Canine Data Commons (ICDC) to make data from studies of spontaneously-arising cancer in pet dogs accessible for analysis, enabling hypothesis-driven comparative analysis of canine and human data.

Results: In recognizing the importance of a comparative oncology research approach, DCTD has launched initiatives to expand the value, use, and impact of spontaneously-arising cancers in pet dogs including: (1) Cancer Center Support Grant (P30) supplements to sequence canine cancers including some that affect children (glioma, lymphoma, osteosarcoma) and (2) UO1/U24 awards to study immunotherapies in canine cancer clinical trials. Data currently in the ICDC include: a small molecule clinical trial in dogs with lymphoma, a transcriptomic study of multiple tumor types, and a genomic study of glioma.

Conclusion: ICDC anticipates adding pathology and imaging data and, as part of the NCI Cancer Research Data Commons, will enable veterinary oncology researchers to access and analyze data from other repositories, such as the Genomic Data Commons (GDC), to further relate their findings to those of humans. ICDC was publicly announced on National Dog Day, August 26, 2020. Projects are now being accepted at https://caninecommons.cancer.gov.

Funding Information: Funded by NCI Contract No. HHSN261201500003I Matthew Beyers, Philip Musk, Mark Jensen, Amit Mukherjee, John Otridge, Ralph Parchment, Biomedical Informatics and Data Science Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702

128 FELINE CUTANEOUS LYMPHOMA: A RETROSPECTIVE REVIEW OF PRESENTATION, DIAGNOSIS, AND RESPONSE TO TREATMENT

Presenter Name: Jacob Siewert Presenter Institution: Colorado State University Presenter Email: [email protected]

Authors: Siewert, Jacob1; Pellin, Mackenzie 2; Husbands, Brian3; Scavelli, Diane1; Curran, Kaitlin 4 1Colorado State University 2University of Wisconsin 3Ohio State University 4Oregon State University

Introduction: Objective: To further characterize the clinical presentation of feline cutaneous lymphoma with a secondary goal of evaluating response to treatment.

Methods: Veterinary oncologists at four academic veterinary hospitals submitted cases of feline patients with cutaneous lymphoma diagnosed by histopathology or cytology. Signalment, FeLV/FIV status, physical examination findings, clinical signs, diagnostic tests, therapy, response and outcome, and necropsy findings, when available, were recorded.

Results: Forty-one patients were identified and described. The majority of patients were domestic shorthairs (n=29) with a mean age at diagnosis of 11.6 years. Males were over-represented in the population (n=30). FIV/FeLV infection was rare, although in a majority of patients (n=33) the FIV/FeLV status was unknown. Thirty-six patients were treated with a variety of modalities, including surgery, radiation, CCNU, doxorubicin, cyclophosphamide, CHOP, and prednisolone only. In multiple patients, surgery was combined with a systemic therapy. Of 36 patients treated with some form of therapy, 18 responded (p=0.004) with seven patients achieving a PR and 11 patients achieving a CR. Response to therapy appeared associated with survival; however, this population was highly censored. Immunophenotype, presence of epitheliotropism, and cell size did not influence treatment response.

Conclusion: Physical examination findings varied among patients. No relationship between immunophenotype, presence of epitheliotropism, and/or cell size was and response to treatment was found, but this was severely limited by small numbers of patients and heterogeneous treatment protocols. Further studies are necessary to evaluate the effect of specific treatment modalities on prognosis.

129 ANALYSIS OF CANINE MYELOID DERIVED SUPPRESSOR CELLS (MDSCS) UTILIZING FLUORESCENCE-ACTIVATED CELL SORTING, RNA PROTECTION MEDIUMS TO YIELD GOOD QUALITY RNA FOR SINGLE CELL ANALYSIS USING THE 10X GENOMICS PLATFORM

Presenter Name: Kirsten Jackson Presenter Institution: University of Florida Presenter Email: [email protected]

Authors: Jackson, Kirsten1; Doty, Andria1; Hutchison, Shana1; Cortes-Hinojosa, Galaxia1; Sahay, Bikash1; Lejeune, Amandine1; Bechtel, Sandra1; Milner, Rowan1 1University of Florida

Introduction: Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for isolation of specific cell populations that can then be further evaluated by Next Generation Sequencing. When utilizing FACS for population isolation prior to NextGen sequencing, it is important to consider the protection of RNA from RNase activity, environmental conditions, and the sort efficiency to ensure optimum sample quality.

Methods: Canine Myeloid Derived Suppressor Cells (MDSC) were isolated from peripheral blood samples of healthy canines and oral melanoma patients. Multiple techniques were utilized for the protection of RNA prior to, during, and after FACS. After ideal RNA quality was obtained, single-cell analysis using the 10x Genomics platform was performed on a population of sorted cells from a healthy canine and a melanoma patient.

Results: A combination of RNAlater ™ and an RNase inhibitor (RiboLock™) was found to provide high-quality RNA from FACS sorted canine MDSCs. Enrichment of the rare cell population also improved the sorting efficiency and dramatically reduced the time needed for sorting. Single-cell analysis was carried out and provided good quality RNA that is currently undergoing further analysis.

Conclusion: Overall, there are multiple techniques that can be used to protect RNA prior to NextGen sequencing and a combination of these techniques is recommended to provide the best information from samples while minimizing gene changes due to stress and degradation. As shown here, accounting for protection of RNA allowed for isolation of good quality RNA that allowed for sample evaluation at a single cell level.

Funding Information: This work was supported by a resident research grant from the University of Florida, College of Veterinary Medicine and donated funding from the Olive’s Way Foundation and the Milner Comparative Oncology Lab

130 PILOT TRIAL OF DOSE-INTENSE COMBINATION CHOP CHEMOTHERAPY IN LABORATORY DOGS

Presenter Name: Rothman Reyes Presenter Institution: Purdue University Presenter Email: [email protected]

Authors: Reyes, Rothman1; Childress, Michael1; Tullius, Jeri1; McCain, Robyn1 1Purdue University

Introduction: CHOP chemotherapy remains the standard treatment for canine multicentric lymphoma but is rarely curative. In contrast, CHOP cures approximately 30–40% of humans with diffuse large B-cell lymphoma. This difference in outcomes may be partially explained by the greater dose intensity of human CHOP protocols.

Methods: To increase the dose intensity of canine CHOP protocol without significantly augmenting toxicity, we used the principle of summation dose intensity (SDI) to derive a novel combination CHOP chemotherapy protocol that models human dosing schedules. Our hypothesis was that the new protocol would be well tolerated when given to healthy mixed-breed laboratory dogs. Three dogs >1 year of age and weighing >20 kg were enrolled in a 42-day pilot trial. All dogs received cyclophosphamide (62.5 mg/m2 PO), vincristine (0.35 mg/m2 IV), and doxorubicin (7.5 mg/ m2 IV) on day 1, and prednisone (50 mg/m2 PO) on days 1-5. On day 22, all dogs received escalated dosages of cyclophosphamide 83.3 mg/m2 PO, vincristine 0.47 mg/m2 IV, and doxorubicin 10 mg/m2 IV. Prednisone was given at 50 mg/m2 PO on days 22-26. Antiemetic and antimicrobial prophylaxis was provided to all dogs.

Results: Treatment-related adverse events were mild and responsive to supportive care. All dogs developed grade 2 (2) or 3 (1) neutropenia 10 days after the second treatment, all of which resolved within 72 hours.

Conclusion: The excellent tolerability of this novel CHOP chemotherapy regimen in laboratory dogs supports efficacy testing in cancer-bearing dogs.

Funding Information: Purdue University Center for Cancer Research

131 EXPLORING THE ASSOCIATION OF INTRATUMORAL IMMUNE CELL INFILTRATES WITH HISTOPATHOLOGIC GRADE IN CANINE MAST CELL TUMORS

Presenter Name: Victoria Costa Presenter Institution: Louisiana State University School of Veterinary Medicine Presenter Email: [email protected]

Authors: Costa, Victoria1; Soileau, Aimee1; Carossino, Mariano1; Langohr, Ingeborg1; Balasuriya, Udeni1; Withers, Sita1 1Louisiana State University School of Veterinary Medicine

Introduction: Canine mast cell tumors (cMCTs) vary in their biological behavior, treatment, and prognosis, based on their grade. Immune cell infiltration has been associated with prognosis and response to treatments in some human cancers, and immune-targeting therapeutics are increasingly being explored in veterinary oncology. However, currently little is known about the immune microenvironment in low- and high-grade cMCTs. Therefore, the objective of this study was to determine the prevalence of T lymphocytes, T regulatory lymphocytes, and macrophages, in low- and high-grade cMCTs.

Methods: The Louisiana Animal Disease Diagnostic Laboratory (LADDL) database was searched for cases of cMCTs. Twenty-five low-grade and 25 high-grade formalin-fixed paraffin-embedded cMCT samples were identified. Immunohistochemistry (IHC) was performed to detect CD3, FoxP3, and Iba1 on sequential sections. Three 400x fields with the highest numbers of CD3+ cells were identified for each tumor. The percentage of CD3+, FOXP3+, and Iba1+ cells was quantified in each of these three “hot-spot” fields using ImageJ software.

Results: Immune cell infiltrates in five high-grade and four low-grade cMCTs have been quantified so far. Numerically higher average

Conclusion: T lymphocytes and macrophages may be increased in high-grade, compared to low-grade cMCTs, although analysis of remaining samples will be needed to determine if statistical significance exists between groups. Increased immune infiltrates in high-grade tumors may indicate that the immune microenvironment plays a role in the aggressive behavior of high-grade cMCTs.

132 PROGNOSTIC SIGNIFICANCE OF KI67, AGNOR, CKIT PCR, AND KIT IHC FOR COMPLETELY EXCISED LOW GRADE CANINE MAST CELL TUMORS.

Presenter Name: Alex Pyuen Presenter Institution: Colorado State University Presenter Email: [email protected]

Authors: Pyuen, Alex1; Regan, Daniel1; Avery, Anne1; Thamm, Douglas1 1Colorado State University, Fort Collins, CO

Introduction: Mast cell tumors account for approximately 20% of skin tumors in dogs and carry a wide range of biologic behavior. There are many proposed mechanisms of determining mast cell tumor prognosis, including several marketed “prognostic panels” which use histologic criteria, immunohistochemistry, and molecular tests to predict prognosis and guide treatment. The aim of this study is to retrospectively evaluate if these tests (Ki67, AgNOR, cKit PCR, and KIT IHC) provide prognostically significant information with regards to progression free interval and overall survival for completely excised low grade mast cell tumors in dogs.

Methods: A medical records search was performed from March 2018 and prior for cases with completely excised low- grade mast cell tumors treated with surgery alone for which histology blocks were still available. Ki67, AgNOR, cKit PCR, and KIT IHC were submitted on all histology samples and medical records were used to evaluate for disease recurrence and/or metastasis for the calculation of progression free interval and for overall survival.

Results: At the time of abstract submission 53 cases for a total of 67 tumors met the inclusion criteria. The majority were mixed breed dogs with a median age at diagnosis of 7.8 years. Median follow up time was 1.6 years. There were three reports of confirmed or suspected local recurrence and 18 instances of confirmed or suspected de novo mast cell tumors at a distant cutaneous site.

Conclusion: Prognostic significance of Ki67, AgNOR, cKit PCR, and KIT IHC is pending and will be available prior to presentation.

Funding Information: None.

133 PROGNOSTIC SIGNIFICANCE OF COMPLETE RESPONSE DURING RADIATION IN FELINE SINO-NASAL LYMPHOMA: A RETROSPECTIVE COHORT STUDY

Presenter Name: Jacqueline Bowal Presenter Institution: Ontario Veterinary College Presenter Email: [email protected]

Authors: Bowal, Jacqueline1; Poirier, Valerie1; Vail, David2 1Mona Campbell Centre for Animal Cancer, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada 2School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI

Introduction: Radiation therapy (RT) is the treatment of choice for feline sino-nasal lymphoma (fsnLSA) and the rate of local response during RT is not defined. Approximately 20-30% will develop systemic progression within one year. Our hypothesis was that the majority of cats will be in complete remission (CR) at the end of RT and that cats in CR will have longer progression-free survival (PFS).

Methods: Single institution retrospective cohort study. Inclusion criteria were histologically confirmed fsnLSA, negative stage, treated with RT only (10 x 4.2 Gy) and at least 1-year follow-up. Tumor volumes were measured on the first and last RT cone-beam CT (CBCT). CR was defined as >90% reduction in volume. PFS was defined from first RT to systemic/local progression or death. Kaplan Meier analysis was performed and cats were divided based on end of RT response: CR versus non-CR.

Results: Thirteen cats met the inclusion criteria. By the end of RT, 7 (54%) were in CR. Overall median PFS was 660 days. The median PFS of non-CR was significantly greater (727 days) compared to CR (149 days), hazard ratio: 11 (95% CI: 2.3-51.6), (p=0.003). At 1-year post RT, 6/7 (86%) of CR had progressed versus 0/6 (0%) of non-CR. Four (30%) developed systemic LSA and 2 (15%) had local recurrence.

Conclusion: The outcome of this cohort of cats is the opposite of our expectation, suggesting that CR by the end of RT would be a negative prognostic factor. Further investigation with a larger cohort of cats is necessary to confirm this finding.

134 CYCLICAL 10-DAY DOSING OF MELPHALAN FOR THE TREATMENT OF CANINE MULTIPLE MYELOMA

Presenter Name: Lucy Teddy Presenter Institution: Cornell University Presenter Email: [email protected]

Authors: Teddy, Lucy1; Hume, Kelly1 1Cornell University

Introduction: Canine multiple myeloma (MM) is typically treated with melphalan chemotherapy. A protocol with repeated cycles of melphalan dosed “10 days on 10 days off” (10/10) has been used anecdotally at our institution but has not been described in the literature. Our objectives are to use a retrospective case series to describe the outcome and adverse events of the 10/10 protocol and to compare our findings to other protocols reported in the literature. We hypothesize that the 10/10 protocol will have similar outcomes compared to other published protocols.

Methods: Dogs diagnosed with MM that received melphalan treatment at Cornell University Hospital for Animals were identified via a database search. Case records were retrospectively reviewed. Data extracted included signalment, clinical signs prior to diagnosis, staging tests, chemotherapy treatments, response to treatment, duration of treatment, length of follow up, adverse events, and outcome.

Results: Seventeen dogs (4 females, 13 males) met the inclusion criteria. Median age was 10 years old and median body weight was 32 kg. The most common presenting clinical sign was lethargy/weakness. Bone marrow aspiration and analysis occurred in 16/17 dogs. Complete remission was defined by a normalized globulin concentrated and was achieved in 10/17 dogs (59%) with a median time to remission of 39 days. Median progression free survival was 651 days.

Conclusion: Outcome with the 10/10 protocol is similar to that reported for daily melphalan dosing, but lower compared to pulse dosing. Data on prognostic factors and adverse events are still being analyzed for significance and will be presented.

Funding Information: No funding was used for this project.

135 RESPONSE TO RADIOACTIVE IODINE (I131) ADMINISTRATION IN DOGS WITH THYROID CARCINOMA

Presenter Name: Selvi Jegatheeson Presenter Institution: University of Melbourne Presenter Email: [email protected]

Authors: Jegatheeson, Selvi1; Zuber, Max2; Woodward, Andrew1; Cannon, Claire1 1University of Melbourne 2Gladesville Veterinary Hospital

Introduction: Responses rates and duration of control of canine thyroid carcinomas treated with radioactive iodine (I131) have not previously been described. This study aimed to describe tumour response and progression-free interval (PFI) in dogs with thyroid carcinomas treated with I131. Secondary aims were to describe the overall survival time (OST) and identify prognostic factors.

Methods: A retrospective review of records from two institutions identified 66 dogs with thyroid carcinoma treated with I131 from January 2010 – April 2020. Response was described using RECIST, or a subjective response assessment in cases without tumour measurements. Univariable and multivariable analysis was performed to identify prognostic factors.

Results: Forty-eight dogs (72.7%) were treatment-naïve and 18 dogs (27.3%) had prior therapy. RECIST responses were available for 33 dogs and subjective responses for 59 dogs. The overall response rates (ORR) were 35.3% (3 complete and, 9 partial responses) with RECIST, and 23.7% with subjective assessment (4 complete and 10 partial responses). Improvement in clinical signs was seen in 27 of 37 dogs (70.3%). Kaplan-Meier estimated overall median [95% confidence interval] PFI was 305 [249-578] (n= 53), and OST was 616 [441-867] days (n = 55). Dogs treated with prior therapy had a longer OST than treatment naïve dogs, and responders had a longer PFI and OST than non- responders.

Conclusion: Radioactive I131 can provide local tumour control in a proportion of dogs. Thyroidectomy prior to I131 may improve outcomes.

Funding Information: No funding was used for this project.

136 PHARMACOKINETICS AND TOXICITY OF CANNADROPS™ COCONUT, A COMMERCIALLY AVAILABLE CBD OIL FORMULATION IN LABORATORY DOGS

Presenter Name: Luis Lembcke Presenter Institution: Purdue University Presenter Email: [email protected]

Authors: Lembcke, Luis1; McCain, Robyn2; Jannasch, Amber2; Ogata, Niwako1; Knipp, Gregory3; Fulkerson, Christopher1 1Purdue University, Veterinary Clinical Science 2Purdue University, Bindley Bioscience Center 3Purdue University, Department of Industrial and Physical Pharmacy

Introduction: The deregulation of industrial hemp derived cannabidiol (CBD) in the United States has increased the availability of direct to consumer veterinary CBD products. The purpose of this study was to evaluate the pharmacokinetics and adverse event profile of a commercially available CBD product (CannaDrops™ Coconut, Healthy Hemp Pet Company, Salt Lake City, UT, USA) in healthy laboratory dogs.

Methods: Three purpose-bred laboratory dogs were used in this study. Pharmacokinetic parameters were determined using blood samples collected at 0.5, 1, 2, 3, 4, 8 and 12 hours following an oral dose of 2 mg kg-1. The maximum tolerated dose (MTD) was determined following dose escalation from 2 to 12 mg kg-1 PO q 12 hrs.

Results: The mean tmax was 2.3 ± 0.47 h, mean t1/2 was 2.4 ± 0.20 h, and mean Cmax was 143.2 ± 88.9 ng ml−1. The dose limiting toxicity (DLT) was VCOG-CTCAEv1.1 grade 3 diarrhea. The MTD was 10 mg kg-1 PO q12 hours. Diarrhea resolved in all three dogs with supportive care. ALP was elevated from baseline in all three dogs and returned to normal after CBD was withdrawn. Following chronic MTD dosing (10 mg kg-1 PO q 12 hrs), the mean plasma concentration at 2 hours post dose was 1061.1 ± 267.2 and 159.4 ± 104.1 ng ml−1 for CBD and 7-OH-CBD, respectively.

Conclusion: To the authors’ knowledge, this is the first report of detection of 7-OH-CBD, a purported active CBD metabolite, in the dog. This formulation of CBD was well tolerated up to the MTD and further investigation of its activity in pet dogs with cancer is warranted.

Funding Information: Salary support for Dr. Fulkerson was provided as part of funding by Healthy Hemp Pet Company. However, this company did not have any input on study design or approval of the abstract/manuscript.

137 THE IMPORTANCE OF IMMUNOHISTOCHEMISTRY IN THE DIAGNOSIS OF SUSPECTED NON-VISCERAL LEIOMYOSARCOMA

Presenter Name: Rachel Brady Presenter Institution: UC Davis School of Veterinary Medicine Presenter Email: [email protected]

Authors: Brady, Rachel1; Choi, Eunju1; Rebhun, Robert1; Skorupski, Katherine1; Burton, Jenna1; Willcox, Jennifer1 1University of California, Davis School of Veterinary Medicine, Davis, CA

Introduction: The objective of this study was to describe the histopathological and immunohistochemical (IHC) characteristics of non-visceral leiomyosarcoma in dogs.

Methods: A retrospective case series was performed of 22 client-owned dogs. Medical records were reviewed to identify dogs originally diagnosed with non-visceral leiomyosarcoma. Histologic and clinical details were obtained. Biopsy samples were reviewed by a single pathologist using standardized histologic criteria including appearance and IHC labeling (positive smooth muscle actin (SMA) and desmin or positive SMA and laminin).

Results: Twenty-two possible non-visceral leiomyosarcomas were identified. Most dogs were middle aged (median 8.6 years, range 0.9 to 14.6) with variably located subcutaneous and cutaneous masses. Of the 22 cases, only four were consistent with a definitive diagnosis of non-visceral leiomyoma (1) or leiomyosarcoma (3) based on the established criteria. Of these, only the leiomyoma had diffuse immunoreactivity against all three markers, while all three leiomyosarcomas had diffuse SMA and near diffuse laminin staining. Eleven additional samples had moderately consistent histologic features but variable immunoreactivity, and a definitive diagnosis could not be determined. Interestingly, five samples were reconsidered as benign soft tissue lesions. Two samples were reclassified as fibrosarcoma.

Conclusion: These data highlight the uncommon nature of non-visceral leiomyosarcoma and the importance of immunohistochemical analysis for diagnosis of sarcoma subtypes. SMA is a common single IHC used for the diagnosis of leiomyosarcoma, but standardized IHC profiles are lacking for classification of smooth muscle tumors. Further research is required to clarify the histologic and immunohistologic features and the clinical significance of non-visceral SMA positive spindle tumors

Funding Information: Center for Companion Animal Health, UC Davis School of Veterinary Medicine resident grant

138 Technician Program and Speaker Notes

Sponsored by

139 Technician Workshop

It is with great pride that we welcome you to the 2020 Veterinary Cancer Society’s Technician Workshop presented on a virtual platform. Due to the virtual nature of this year’s workshop, the schedule has been altered. Lectures will be available both live and on-demand, with many presenters at the ready to answer your questions. Although we cannot meet in person, our goal is to provide you with as much of a interactive experience as possible! This year’s workshop opens on Thursday evening with a presentation Aspen Schreiner, CVT Casey Mobley, CVT from Amatheon Pharmaceuticals. Following this will be the ever-popular Onology Technician Onology Technician cytology lab presented again by Drs. Davis Seelig and Daniel Heinrich of the University of Minnesota. The workshop continues on Friday with a presentation from one of our fabulous sponsors, Epicur Pharma. We will then dive into our keynote presentation. Kara Burns, MS, MEd, LVT, VTS (Nutrition) will discuss nutrition and the cancer patient. With growing client awareness of the importance of nutrition, this lecture will help give technicians a better understanding of nutrition in the cancer patient, and will provide tools to help guide clients in dietary choices. The workshop offers advanced and beginner tracks on Friday. These topics have been tailored to fit the needs of all types of oncology technicians and will include discussions on chemotherapy side effects and extravasation, paraneoplastic syndromes, lymphoma versus leukemia, molecular diagnostics, cryosurgery, and electrochemotherapy. Speakers for the day include expert technicians and doctors. Other Friday highlights include a presentation by Elias Animal Health, as well as a lecture on chemotherapy safety. We finish out the day with our radiation therapy workshop, presented by radiation oncologist Dr. Kelsey Pohlmann. We are both very excited for this workshop which will include discussion of the “ins and outs” of radiation therapy - a sort of “technician’s guide to radiation therapy”, if you will. Our final day of the workshop will include a Technician case report on feline gastrointestinal lymphoma presented by Racheal Rail, RVT, as well as case studies presented by our friends at Best Pet Rx. We will continue with a schedule packed full of varying lecture topics and dynamic speakers to include (but certainly won’t be limited to) cancer grading and staging, oncology emergencies, anesthesia and analgesia, and treatment of mast cell tumors. The closing session will dive into a skill necessary to all veterinary oncology technicians – communication through grief. Danielle DeCormier, LVT, VTS (Oncology), CCFP, will lead our discussion on how to effectively communicate with clients during their pet’s journey through cancer. After this enlightening lecture, be sure to bring your “happy hour treat” and join us for an informational, LIVE discussion on applying for your VTS oncology license led by your Technician Liaison, Brooke Quesnell, CVT, VTS (Oncology). The conference concludes at 7:30 p.m. with our Closing Event, Awards and Membership Meeting. We are very excited to be hosting such a wide variety of expert speakers, as well as lecture topics designed specifically based off of feedback from YOU! While we are unable to meet in Daytona Beach, we are still looking forward to this educational, exciting, and well-rounded conference. We hope you are, too! Aspen Schreiner, CVT and Casey Mobley, CVT Program Co-Chairs

140 Technician Workshop Schedule

Thursday, October 15, 2020 TIME TOPIC PRESENTER PAGE 5:00 pm Virtual Exhibit Hall and Poster Hall Open 5:30 pm - 5:45 pm Conference Welcome & Virtual Conference Details 5:45 pm - 6:15 pm Sponsor Presentation by Amatheon Animal Health TBD 6:15 pm - 8:30 pm Diagnostic Veterinary Cytology for the Veterinary Dr. Davis Seelig & 143 Technician Dr. Daniel Heinrich Friday, October 16, 2020 TIME TOPIC PRESENTER PAGE 9:00 am - 10:00 am Keynote Presentation: Cancer Cachexia - When all Kara Burns 144 your patients can eat is themselves 10:00 am - 10:30 am BREAK/EXHIBIT HALL/POSTER VIEWING 10:30 am - 11:00 am Sponsor Presentation by Epicur Pharma Learn the difference: Manufactured versus compounded 11:00 am - 11:50 am Increasing the veterinary nurse’s role through Dr. Shea Cox 148 palliative care 11:50 am - 12:35 pm LUNCH BREAK/EXHIBIT HALL/POSTER VIEWING Beginning Track: Most appropriate for technicians with limited oncology experience. Advanced Track: Most appropriate for technicians with advanced oncology experience. 12:35 pm - 1:25 pm Beginning Track: Chemo side effects, extravasation Samantha Fritz 149 Advanced Track: Paraneoplastic syndromes Casey Foster (Mylet) 152 1:30 pm - 2:20 pm Beginning Track: Lymphoma vs. Leukemia Kim Albin 153 Advanced Track: Molecular diagnostics Dr. Anne Avery 155 2:20 pm - 2:40 pm BREAK/EXHIBIT HALL/POSTER VIEWING 2:40 pm - 3:30 pm Beginning Track: Cryosurgery: A super COOL Aspen Schreiner 156 treatment option Advanced Track: Electrochemotherapy Brooke Quesnell 157 3:30 pm - 4:00 pm BREAK/EXHIBIT HALL/POSTER VIEWING 4:00 pm - 4:50 pm Chemotherapy safety & handling Jenny Fisher 160 5:30 pm - 6:00 pm Sponsor Presentation by Elias Animal Health Adoptive T cell therapy: Harnessing a dogs own immune system to fight cancer 6:00 pm - 8:15 pm Implementing State-of-the-Art Radiation Therapy in Dr. Kelsey Pohlmann 161 Veterinary Practice

This conference takes place in Eastern Time.

141 Technician Workshop Schedule

Saturday, October 17, 2020 TIME TOPIC PRESENTER PAGE 10:15 am - 11:05 am Delving into diagnostic imaging Madeline Ederer 168 11:15 am - 11:45 am Sponsored Presentation by Best Pet Rx Customized solutions for supportive therapy in oncology patients – Case overviews 11:45 am - 12:00 pm Case Report: Rachael Rail 169 Giving up the commitment to the probable: Durable remission in a case of feline large cell GI lymphoma 12:00 pm - 12:45 pm LUNCH BREAK/EXHIBITS/POSTER VIEWING 12:45 pm - 1:35 pm Cancer grading & staging: Understanding the basics Dr. Zachary Neumann 171 1:40 pm - 2:30 pm Oncology emergencies Dr. Andi Flory 172 2:40 pm - 3:30 pm Mast cell tumors - What your mother didn’t tell you Dr. Pam Jones 174 3:30 pm - 4:00 pm BREAK/EXHIBITS/POSTER VIEWING 4:00 pm - 4:50 pm Anesthesia and analgesia for oncology patients Nicole Shuey 176 5:00 pm - 6:00 pm Technician or therapist: Communication through grief Danielle DeCormier 179 6:40 pm - 7:10 pm How to become a VTS Brooke Quesnell 7:30 pm - 8:30 pm Closing Event, Awards and Membership Meeting

This conference takes place in Eastern Time.

142 Technician Workshop Featured Speakers

Diagnostic Veterinary Cytology for the Veterinary Techician Thursday, October 15, 2020 | 6:15 pm – 8:30 pm Cytology is among one of the most common diagnostics in primary and specialty care veterinary hospitals. The veterinary technician can play a significant and important role in diagnostic microscopy, including sample collection, sample preparation and quality assurance, and sample analysis. This session will focus on techniques to maximize the diagnostic value of a sample and provide instruction Dr. Davis Seelig Dr. Daniel Heinrich on the microscopic interpretation through the use of representative case examples. In addition to cytology, the session will include a review of ancillary tests for lymphoid neoplasms (lymphoma and leukemia), including flow cytometry and PARR (lymphoma PCR) testing. Samples will include cytology preparations and instruction will be provided by two board-certified veterinary clinical pathologists. Keynote Presentation: Cancer Cachexia - When all your patients can eat is themselves Friday, October 16, 2020 | 9:00 am – 10:00 am Cancer is a common disease of dogs and cats. Advances in cancer therapy have resulted in increased survival time for many veterinary patients. Despite these advances, malnutrition remains a common problem in these patients. Cancer-associated malnutrition occurs as a consequence of an imbalance between the nutritional needs of the patient, the demands of the tumor, and the availability of nutrients in the body. Kara M. Burns, MS, MEd, Cachexia is a specific form of malnutrition characterized by loss of lean body mass, LVT, VTS (Nutrition) VTS-H muscle wasting, and impaired immune, physical, and mental function. Cancer cachexiais (Internal Medicine, Dentistry) a clinically important problem in dogs and cats. The healthcare team must be able to identify cachexia and begin proper nutritional management as early as possible. Implementing State-of-the-Art Radiation Therapy in Veterinary Practice Friday, October 16, 2020 | 6:00 pm – 8:15 pm The purpose of this presentation is to provide a general overview of radiation therapy in veterinary medicine and will provide a brief overview of basic physics and biology of external beam radiation. We will also discuss the evolution of radiation therapy and how it has impacted our ability to treat cancers that were previously deemed untreatable. We will discuss indications for referral, reviewing commonly treated tumor types and evidence- Dr. Kelsey Pohlmann based outcomes that support the use of stereotactic radiation in veterinary medicine. Finally, we will define the role of the veterinary technician in implementing radiation therapy in veterinary practice. This conference takes place in Eastern Time.

143 CANCER CACHEXIA: WHEN ALL YOUR PATIENTS CAN EAT IS THEMSELVES Kara M. Burns, MS, MEd, LVT, VTS (Nutrition) Academy of Veterinary Nutrition Technicians

Cachexia is a multifactorial syndrome characterized by progressive weight loss that is oftentimes is accompanied by anorexia.1 Unlike weight loss seen with starvation or anorexia, cachexia is distinguished by a loss of adipose tissue with accompanying loss of lean body mass, primarily muscle. Non-muscle protein as found in the organs is preserved in cachexia but not in starvation. Significant loss of mineral content in the bones can also be seen contributing to the overall weakness found in many patients.1 This discussion will take a look at cachexia and the role nutrition plays in managing cachexia.

STARVATION VS. CACHEXIA Disease associated weight loss differs from that seen with simple starvation. In cachexia there is an equal loss of muscle and fat characterized by increased catabolism of skeletal muscle. During starvation, fat is mobilized first sparing muscle proteins, resting energy expenditure is decreased and glucose metabolism is reduced. In contrast, cachectic patients have normal or elevated resting energy expenditures and glucose turnover. Adequate nutrition will halt and reverse the metabolic alterations that accompany simple starvation but will not completely reverse the metabolic disturbances associated with cachexia.

This loss of lean body mass has a harmful effect on strength, immune function and overall survival.2 Weight loss provides an important prognostic indicator in overall survival of the patient. Increased weight loss is inversely proportional to survival time.1,3 Loss of muscle mass is first noticed over the epaxial, gluteal, scapular, and temporal muscles.4 and is easily detected during a routine physical exam. There does not appear to be a cause and effect relationship between anorexia and cachexia, with weight loss exceeding that expected with simply and decrease in caloric intake.1

Cachexia has been seen in animals with cancers, cardiac disease, renal disease and other serious illnesses and injuries. Loss of 25-50% of the lean body mass compromises the immune system and affects muscle strength, with death resulting from infections, pulmonary failure or both .5

PATHOPHYSIOLOGY Cancer associated starvation occurs as a consequence of an imbalance between the nutritional needs of the patient, the demands of the tumor and the availability of nutrients in the body.6,7 The competition for nutrients between the tumor and the host promotes a variety of metabolic disturbances including alterations in carbohydrate, lipid and protein metabolism. Cytokines play a key role as the main humoral and tumor derived factors involved in cancer cachexia and may be responsible for the majority of metabolic changes associated with cancer cachexia. Table 1 summarizes the effects of cytokines on nutrient metabolism in patients with cancer cachexia.6 Table 1. CHO Increased resistance to insulin Increased glucose synthesis Increased Cori cycle activity Protein Increased protein breakdown (catabolism) Increased liver (acute phase proteins) and tumor protein synthesis Lipid Increased lipid mobilization Elevated levels of triglycerides

PHASES OF CACHEXIA There are three phases of cachexia identified in humans; the veterinary profession manages cachexia based on these patterns. Throughout the first phase, the patient does not exhibit any clinical signs; however, the biochemical changes are already occurring. These include elevations seen in lactate levels through the glycolysis process, an increase in insulin levels, causing peripheral insulin resistance, and alterations in amino acid and lipid profiles.3

144 During the second phase of cachexia, clinical signs can become evident presenting as anorexia, weight loss, and lethargy. Many owners attribute these early signs to the pet’s aging and may not recognize the clinical significance. The final phase is characterized by marked loss of body fat and protein stores severe debilitation, weakness and biochemical evidence of negative nitrogen balance.3 If left untreated cachexia can be the ultimate cause of death.

THERAPEUTIC STRATEGIES The optimal therapy is to manage the underlying disease process. In veterinary medicine therapeutic strategies generally include management of anorexia, nutritional support / supplementation, and omega-3 fatty acids.

MANAGING ANOREXIA / HYPOREXIA Nutritional counseling should be a part of the pretreatment plan for all patients. Veterinary technicians should help owners understand the importance of measuring intake and should have a sequential plan for maintaining nutritional support throughout the treatment process. This may include simple strategies to increase consumption initially (see table) which may progress to assisted feeding if necessary. In a patient with a functional GI tract enteral feeding is preferred. Strategies for managing anorexia in dogs and cats have been thoroughly reviewed.8 Table 2 is a brief review of those recommendations.

Table 2. Create ambiance /improve Create a quiet comfortable feeding area away from disturbances. service Increase Palatability Increase moisture Switch to canned food / add water to kibble. Caution: some cats may have texture preferences and actually prefer dry food Increase fat Higher fat diets are more energy dense so less food may be required to meet needs. Increase protein Dogs may select foods with higher protein level Sweet and salty Adding sweet flavor (No artificial sweeteners) as top dressing may increase palatability for dogs but not cats. Adding salt (if appropriate) may increase palatability for dogs but not cats. Freshness, aroma, and food Provide ‘fresh’ canned / dry foods or home prepared foods. Warming foods to temperature no greater than body temperature may also release aromas. Rarity Uncommon or rare food may entice some dogs and cats to eat. Best to offer foods that are uncommon but not completely novel. Variety If a variety of options are provided each should be carefully measured to ensure accurate accounting of food intake /preferences. This approach should not be used if food aversions are likely, so that the patient will not develop a learned aversion to all of the therapeutic options. Drug administration Never top dress food with medication or use the desired diet to administer medication. Avoid giving medications immediately before/ after a meal if possible. Eliminate physical Be sure food bowls are accessible and account for any limitations in barriers movement (raise bowls, remove E collars etc) Appetite Stimulating No recommended as effects are unpredictable, intermittent and short lasting. drugs If above approaches are ineffective assisted feeding is recommended.

145 KEY NUTRITIONAL FACTORS IN THE MANAGEMENT OF CACHEXIA While it is not possible to reverse the wasting process with nutritional supplements alone, our patients do need to be fed, and through manipulation of nutrients benefits can be seen in some of the changes the body is undergoing.1 Proper nutrition can be key to managing cachexia with provision of calories, protein, fat and modulation of cytokine production.2 Specific dietary recommendation should consider the stage of disease, the patient’s energy needs, current and past nutritional status and ability or willingness to eat.3

The caloric distribution in the food should emphasize calories obtained from fats and proteins, rather than carbohydrates since we know that glucose is the preferred fuel for tumor cells in cancer cachexic patients, and fatty acids and amino acids are not. The goal is to feed the patient, and starve the tumor cells. Ideally a food should contain 50-60% of the calories from fat, 30-50% of the calories from protein and the remaining portion from carbohydrates.3,9

Omega-3 fatty acids, especially those found in certain types of fish and fish oil (eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA]), are probably the most important nutraceuticals to consider for animals with cancer.6,9,10 Studies using animal models have shown that supplementation with EPA and DHA can help to prevent cachexia and metastatic disease processes. These fatty acids obtained from the omega-3 class of fats produce less potent inflammatory mediators than the omega-6 class of fatty acids, which are viewed as pro-inflammatory. When the body uses the omega-3 class of fatty acids to produce the cytokines, the inflammatory response produced is decreased in proportion to the level of omega-3 fatty acids in the diet. A recommended a dose of 40 mg/kg of EPA, and 25 mg/ kg of DHA. Using the common formulation of most fish oil capsules, to achieve this concentration you would need to provide ~ 1 gram capsule/10# body weight.2 Many recovery diets already have this incorporated into the diet and additional supplementation is not recommended.

Dietary protein should be highly digestible and go above levels normally used for maintenance of adult animals, due to the catabolic process. Protein levels of 30-50% are recommended, with dogs around 30 to 45% and cats around 4- to 50% DMB. Looking at protein levels another way, the minimum recommend intake is 5.14 grams/100 kcal with 6-7 grams/100 kcal preferred.2,10

As stated previously, tumor cells preferentially use glucose for energy. Selecting a carbohydrate with a lower glycemic index would provide a slower release of carbohydrate generated glucose into the blood stream than would those with higher glycemic indexes. Rice is one of the carbohydrates with a highest glycemic index, with barley, sorghum and corn having much lower glycemic indexes.3

FEEDING METHODS Enteral feeding is always the preferred delivery method, provided the patient has a functional gastrointestinal tract. Oral feeding of a canned or dry pet food would be the first choice with cachexia patients.3,9,10 When a patient is unwilling or unable to consume the desired amount of food orally, various feeding tubes can be used. The specific tube selected will vary based on the patient, the desired length of use and the owner’s willingness to feed at home. Diet selection would also be based on the route selected for feeding. Preparation of home-cooked diets can be used short term to tempt the patient to eat, but should only be used long-term after consultation with a nutritionist. There are also a number of excellent therapeutic diets available that fit the recommended diet profile and can be fed both orally and via tube feeding.

146 SUMMARY It is imperative that veterinary technicians recognize and understand cachexia in patients. It is also necessary for healthcare team members to employ nutritional strategies to help manage anorexia and the imbalance between the nutritional needs of the patient and the availability of nutrients in the body. Feeding strategies should be aimed at alleviating the competition for nutrients between the disease process and the host which result in a variety of metabolic disturbances including alterations in carbohydrate, lipid and protein metabolism. While cachexia is a fairly obvious sign that there are biochemical imbalances within the body, these changes have been occurring for a while by the time we see the physical changes. We need to continue to feed our patients through disease and cancer treatment. By recognizing the changes these processes have caused within the body, our diet recommendation can be used to help the patient rather than the tumor or disease process resulting in more positive outcomes and happier patients and clients.

REFERENCES

1. Tisdale M. Mechanisms of Cancer Cachexia. Physiology Review. April 2009 vol 89 no. 2 pg 381-410 2. Freeman LM, Rush JE. (2012) Nutritional Management of Cardiovascular Diseases in Applied Veterinary Clinical Nutrition (eds Fascetti AJ, Delaney SJ). Wiley-Blackwell Ames, IO. pp 304-307 3. Case L, Daristotle L, Hayek M, Raasch M. (2011) Nutritional care of cancer patients in Canine and Feline Nutrition 3rd ed. Mosby Elsevier Maryland Heights, MO. pp 479-486 4. Freeman LM, Rush JE (2006) Cardiovascular diseases: nutritional modulation in Encyclopedia of Canine Clinical Nutrition (eds Pibot P, Biourge V, Elliott D). Royal Canin Aimargues France EU. pp 321- 336 5. Saker K, Remillard RL. (2010) Critical Care Nutrition and enteral-assisted Feeding in Small animal Clinical Nutiriton 5th ed (eds Hand MS, Thatcher CD, Remillard RL, Roudebush P, Novotny BJ). Mark Morris Institute Topeka KS. pp 441- 442 6. Wortinger A, Burns KM. Nutrition and Disease Management for Veterinary Technicians and Nurses. Wiley Blackwell. 2015: 202-207. 7. Mauldin GE. Nutritional Management of Oncological Diseases. In Applied Veterinary Clinical Nutrition. Fascetti AJ, Delaney SJ, eds. 2012. Wiley-Blackwell, Ames, IA. 8. Delaney SJ. Management of anorexia in Dogs and Cats. Vet Clin Sm An Pract (2006) 36; 1243-1249. 9. Saker, KE, Selting, KA. Cancer. In: Hand MS, Thatcher CD, Remillard RL, Roudebush P, Novotny B. eds. Small Animal Clinical Nutrition, 5th ed. 2010:587-607. 10. Forrester SD, Roudebush P, Davenport DJ. Nutritional Management of the Cancer Patient. In Cancer Management in Small Animal Practice. C. Henry CJ, Higginbotham ML, eds. Elsevier, 2010: 170-176. St. Louis, MO.

Other Reading Resources: - Hemming L, Maher D. Understanding cachexia and excessive weight loss in cancer. Brit J Comm Nursing . (2005) 10 (11); 492-495. - Michel KE, Sorenmo K, Shofer FS. Evaluation of body condition and weight loss in dogs presented to a veterinary oncology service. JVIM (2004) 18; 692-695.

147 A VETERINARY NURSE’S ROLE IN PALLIATIVE CARE Dr. Shea Cox, DVM, CHPV, CVPP

The Role of the Veterinary Nurse in Palliative Care: The Why • HospiceTechnician and palliative care Workshopis, and should be, a nurse-driven service • The need for increased autonomy and evolving roles within the veterinary team • Working Keynoteas a team to improve Speaker care for the client, the patient, and the entire staff

The Role of the Veterinary Nurse in Palliative Care: The How • General considerations:

o Identify a dedicated nurse who can be a palliative care champion for the hospital o Getting hospital “buy in” o Scheduling considerations . Virtually, in-clinic, in-home . Pros and cons of each care delivery model . Recommended length of appointments · The nurse’s role defined, including a detailed discussion around the vital components of each phase of the nursing consult and ongoing case management led by the nurse o Importance of a “chunk and check” approach to client communication and education o The pre-consultation intake . History intake . Veterinary services intake . Evaluation of needs, beliefs and goals intake . Environmental assessment intake o The post-consultation intake . General disease education . Disease trajectories . Pain recognition . Medication education and considerations . Home environment modifications and improvements . End-of-life planning . Grief and pet loss support considerations . Education resources o Reassessments and Check-ins, including recommended frequency based on patient status . Pain reassessment . QOL reassessment . Family goals reassessment

The importance of caring for yourself while caring for your patients

Training opportunities and education resources

148 WHAT KEEPS ONCOLOGY TECHNICIANS UP AT NIGHT: CHEMOTHERAPY COMPLICATIONS AND SIDE EFFECTS Samantha Fritz, CVT, VTS (Oncology)

Introduction Chemotherapy complications and side effects differ with each drug and can range from mild to severe. In human oncology, higher dosing and the use of multi-drug protocols can lead to more severe side effects. In veterinary oncology, we focus heavily on quality of life through treatment and therefore aim to reduce the risk of hospitalization or severe, life threatening complications. However, oncologic emergencies can arise, and a properly trained technician can not only reduce risks but could potentially reduce life alternating effects.

Chemotherapy Side Effects Traditional chemotherapy does not discriminate between normal and cancerous cells. It targets rapidly dividing cells, and these include the normal cells in the bone marrow, hair follicles, and gastrointestinal tract. One acronym from human medicine: B.A.G.

Bone marrow suppression is common with use of most chemotherapeutic drugs and can be noted most during the nadir. This is when the blood cell with the shortest half-life, the neutrophil, has its count dip to its lowest point. While the nadir with many drugs falls in the range of 5-10 days after treatment, it is important to note that different chemotherapy drugs have different chemotherapy nadirs. Carboplatin for example has a nadir at 10 – 14 days post, but a second “double” nadir can be observed at 21 days post. For most chemotherapies, it is important to obtain a complete blood count (CBC) and temperature check at 7 days post to monitor myelosuppression that may occur. During the time of the chemotherapy nadir, though neutropenia is the most critical, thrombocytopenia and anemia may also be noted. Patients with severe neutropenia <1000 neutrophils/μL are at increased risk of sepsis which can lead to death. If a patient presents for a nadir check and is febrile with neutropenia, hospitalization with medical intervention is necessary. If the degree of neutropenia is moderate (1000-1500 neutrophils/uL) or the patient is symptomatic (febrile or lethargic), a preventative PO course of antibiotics may be prescribed to help reduce the possibility of sepsis. For future doses, a dose reduction or complete change of therapy may be indicated.

Alopecia (hair loss), though not life-threatening, can be extremely upsetting to the owner. Alopecia is most commonly noted in the constantly shedding breed dogs, such as poodles and bichon frises. Alopecia is not common with all chemotherapy drugs but is most noted with doxorubicin. Happily, any lost hair does grow back when treatment is completed, though it may grow back a different texture or color.

Gastrointestinal (GI) side effects make up the majority of the chemotherapy side effects that are noted by the owners at home. Most GI side effects are mild and can be self-limiting, but sometimes, if severe enough, medical intervention may be necessary to improve the patient’s quality of life. GI side effects most commonly include vomiting, diarrhea, lack of appetite or nausea. Most chemotherapy GI side effects are noted about 3 – 5 days post. If the owners note any side effects at home, food changes (NPO or a bland diet), the usage of anti-emetics, or anti-diarrhea medications can be used symptomatically, or in a preemptive manner for future treatments if the patient had a history of sensitivities. If the side effects are severe enough to decrease the quality of life of the patient, a dose reduction may be indicated for the next treatment. Though not all patients experience GI side effects, “white feet” breeds such as the herding dogs that express the MDR1 gene mutation may be more susceptible to chemotherapy-induced side effects. Thus, certain precautions may be made when treating such breeds as Australian Shepherds, Collies and even Long-haired Whippets.

Other side effects are less common but can occur with certain chemotherapy drugs. These may be drug- or species- specific and can be severe or even deadly without proper education and awareness. Doxorubicin-induced cardiotoxicity is mostly seen with cumulative doses in dogs, >200 mg/m2. If a patient has cardio insufficiency, abnormal pulses, dilated cardiomyopathy or any atypical rhythms, such as ventricular premature contractions (VPCs), on an electrocardiogram, a cardiology consult with a board-certified cardiologist would be indicated before treatment of doxorubicin. Hepatotoxicity can occur with cumulative dosing of lomustine and therefore preemptive treatment with a liver protectant, such as Denamarin, would be recommended along with monitoring of the liver enzymes. Neurotoxicity can happen with the use

149 of 5-fluorouracil (5-FU) in cats, and therefore is contraindicated and should never be used as it can cause death. Pulmonary toxicity is a fatal side effect with the use of Cisplatin in cats and is therefore contraindicated. Pulmonary fibrosis is a possible complication of Tanovea-CA1 and should be avoided in West Highland White Terriers and other terrier breeds. Neuropathy and gastrointestinal ileus may be noted with frequent vincristine treatments. Sterile hemorrhagic cystitis can occur with IV or long-term usage of oral cyclophosphamide so the use of a diuretic may be recommended to flush out the bladder to avoid this side effect. Asparaginase has the potential of anaphylactic reaction with multiple doses, so therefore, it is recommended to pre-treat with a steroid and antihistamine prior to administration and closely monitor for any symptoms for 24-48 hours post. Asparaginase treatment can also be associated with acute tumor lysis syndrome (ATLS). This is a rare syndrome that mostly affects higher grade lymphoma patients with a heavy tumor burden. The mechanism of asparaginase is to rapidly kill neoplastic cells, which leads to the release of the intracellular components. Patients who experience ATLS may exhibit symptoms hours, or even up to 7 days post administration. Due to the nature of the effects on the body, ATLS can be life threatening if immediate medical intervention is not taken. It is important to note that though ATLS is associated more with Asparaginase treatment in lymphoma/leukemia patients, any patient with a high tumor burden that receives chemotherapy treatment may be at risk.

Extravasation Chemotherapy extravasation is when the injection leaks out of the vessel and into the surrounding tissue. Extravasation can be avoided, but it is imperative to know which drugs are classified as vesicants, and what to do if it occurs. Prevention is key and with any good prevention strategy, knowledge is power. No one plans on chemotherapy extravasation, which is why it’s considered an oncologic emergency, but knowing what to do when it happens can help reduce the severity and may save a life. Unfortunately, most of the extravasation protocols that are in place are based on human medicine due to the lack of research in veterinary oncology.

Technician education is the first and foremost of importance when it comes to chemotherapy administration. Ensuring the staff is properly trained intravenous catheter placement is crucial as a clean stick is required for any type of chemotherapy that is classified as an irritant or vesicant. It is recommended that the chemotherapy administration is supervised by a trained technician so therefore, if there is any possible extravasation, the proper steps to avoid detrimental tissue damage can begin immediately. A basic understanding of the patient’s temperament is also important. The use of pre-medications or sedation may be necessary to ensure the chemotherapy is given safely in patients who are not amendable to safe restraint for administration. A separate room or space where there is less commotion or distraction is recommended for any chemotherapy administration.

Signs of extravasation may be immediate or may arise days or even weeks after the injection. Immediate reactions may include pain at the site, swelling, erythema and inflammation. Severe delayed reactions can include ulcerating tissue necrosis.

Irritants include cyclophosphamide, gemcitabine, and carboplatin. Low vesicant potential drugs include mitoxantrone, 5-FU, dacarbazine and cisplatin. High vesicant potential drugs include mechlorethamine, vincristine, vinblastine, vinorelbine, and doxorubicin.

If any extravasation is noted, the first step is to stop the injection or infusion immediately and pull back as much of the drug as possible. One should NOT flush the catheter. The doctor should be alerted, and notation of the affected area should be made. If there is a bleb of drug, a needle aspiration may be performed to remove excess drug from the subcutaneous space. Note how much of the drug was obtained from the catheter, or from fine needle aspiration of the bleb. A marker should be used to outline the area of extravasation and treatment should start as soon as possible. At home care is similar to standard wound care, as it is crucial that the owner keeps the patient from licking at any extravasation site as it may delay healing, cause infection, or worsen the lesion overall.

For mild vesicant drug extravasation such as cyclophosphamide, cytarabine, carboplatin, and gemcitabine, a dry cold compression should be placed for 20-30 minutes, and then as needed for inflammation. Topical 90% DMSO may also help with carboplatin extravasation. DMSO can be applied to the skin surface every 8 hours for one week in conjunction with anti-inflammatory drugs if necessary.

150 For vinca alkaloid extravasation, a dry warm compress should be applied for 20-30 minutes at a time, for up to 4 times a day for the first 24-48 hours following the extravasation. Hyaluronidase is recommended to help absorb the drug. Human studies show that a 150 U/mL solution of hyaluronidase injected through the existing catheter helped reduce the extravasation severity. For every 1 ml of extravasated drug, 1 ml of hyaluronidase (150 U/mL) was given. Hyaluronidase can also be given subcutaneously, but ensure the needle is changed with every injection. If giving subcutaneously, inject 0.2 ml of the 150 U/mL hyaluronidase around the edge of the extravasation site.

For meclorethamine extravasations, sodium thiosulfate can be injected into the local tissues. To be effective, this should be done immediately. For 10% sodium thiosulfate solution: mix 4 ml with 6 ml preservative-free sterile water for injection. If 25% sodium thiosulfate solution: Mix 1.6 ml with 8.4 ml of preservative-free sterile water for injection. Inject 2 ml for every 1ml of vesicant extravasated through the existing line. Then consider injection 1 ml subcutaneously in 0.1-ml doses clockwise around the site. This can be repeated several times over the next 3 – 4 hours.

For doxorubicin extravasations, apply a dry cold compress immediately for 20-30 minutes at a time 4 times a day for the first 24-48 hours. Dexrazoxane should be administered as a separate intravenous infusion within 6 hours of the extravasation, but sooner administration yields better results. The dexrazoxane should be diluted as instructed, and then additionally diluted to a 1.3-5 mg/ml solution in either 0.9% sodium chloride or 5% dextrose injection. The dose is 1:10 of vesicant:dexrazoxane on day one. Then daily for two additional days. Triple doses have been more effective than a single dose. Surgical intervention may be necessary depending on the severity of the extravasation site. DMSO may be contraindicated in patients that are receiving or are going to receive dexrazoxane for extravasation treatment as a study in mice showed that the DMSO reduced the effectiveness of dexrazoxane. However, other reports in dogs indicate the usage of 90% DMSO without complication, but it is not recommended treatment option alone.

References: 1. “Chemotherapy Extravasation Management.” AAHA Home, www.aaha.org/aaha-guidelines/oncology- configuration/implementation-toolkit/chemotherapy-extravasation-management/. 2. Henry, Carolyn J., and Mary Lynn. Higginbotham. Cancer Management in Small Animal Practice. Elsevier Mosby, 2010. 3. MacDonald, Valerie. “Chemotherapy: Managing side effects and safe handling.” The Canadian Veterinary Journal, June, 2009. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684058/ Accessed 28 July 2020. 4. “Washington State University VCPL - MDR1 Test.” VCPL, vcpl.vetmed.wsu.edu/.

151 PARANEOPLASTIC SYNDROMES – PIECES OF THE PUZZLE Cassandra Foster CVT, VTS Oncology

Paraneoplastic syndromes are an important and often overlooked extension of cancer. Although they are more commonly recognized and studied in human medicine, paraneoplastic syndromes are often present in our veterinary patients as well, and they may cause more morbidity and mortality than the tumor itself. Whether they are life threatening or only threatening to quality of life, paraneoplastic syndromes often require medical intervention, and it is imperative that we recognize and treat them when they occur.

The definition of PNS is “a neoplasm –associated alteration in bodily structure or function that occurs distant to the tumor.”1 Usually caused by substances produced by the tumor, PNS can occur in nearly all body systems. Signs of the PNS can be present months or years before discovery of the primary tumor. Often it is the PNS itself that draws our attention, and because of it, we discover the tumor. Many tumors can cause PNS, and the types of disorders are extremely varied.

GASTROINTESTINAL PNS Cancer Cachexia Gastrointestinal Ulceration

ENDOCRINE PNS Hypercalcemia Hypoglycemia

HEMATOLOGIC PNS Anemia Polycythemia Eosinophilia

NEUROLOGIC PNS Myasthenia Gravis

OTHER PNS Hypertrophic Osteopathy Fever

Paraneoplastic syndromes encompass a wide array of medical conditions all caused by substances produced by tumor cells. Many times, the PNS is the first sign of disease, leading the medical team on a scavenger hunt to find the neoplasia it is hiding. Unfortunately, oftentimes, it is the PNS itself that is the cause of death. Usually, resolution of the primary tumor is the best treatment for the PNS, however adjuvant therapies may be helpful. It is important to recognize the telltale signs of PNS, as they are often the most important pieces of the puzzle.

152 LYMPHOMA VS. LEUKEMIA: BLOOD COUSINS Kim Albin, LVT, VTS (Oncology)

Leukemia and lymphoma are two types of cancer that affect the blood. Both cancers typically affect the white blood cells. Lymphoma is a cancer of a specific white blood cell called a lymphocyte. Leukemia is a broader term describing cancers of blood cell precursor cells and starts within the bone marrow. Pets with lymphoma and leukemia have very similar clinical signs and laboratory test results, and even the most astute pathologist can easily confuse the two diagnoses. The prognosis and treatment options vary greatly, therefore it’s extremely important to have an accurate diagnosis.

Lymphoma Lymphoma is a cancer of lymphocytes, which are a type of white blood cell. There are several different forms of lymphomas in dogs and cats, but the most common form involves an excessive proliferation of lymphoblasts (immature lymphocytes) within lymph nodes and organs of the body. Lymphoma is typically classified as either being of a B-lymphocyte or T-lymphocyte origin.

Leukemia Leukemia refers to several different types of cancers arising from the different blood cell elements within bone marrow. Animals can develop leukemia of white blood cells, red blood cells, or platelets. Acute leukemias are first classified into one of 2 categories: acute lymphoid leukemias (ALL), which arise from immature lymphocytes (and can be of either a B-cell or T-cell origin), and acute non-lymphoid leukemias (also referred to as acute myeloid leukemias or AML), which arise from all other immature blood cell precursors in the bone marrow.

Origins White blood cells are created in the bone marrow via a complicated hierarchy of cell division. Stem cells are the most primitive forms of the blood cell elements. These cells divide and give rise to slightly more specialized cells, which continue to progressively differentiate, until all of the finalized mature blood elements are created and “ready” to be released into the blood stream.

One of the main break off points during the maturation of blood cells in the bone marrow occurs when cells are slated to mature into what are known as lymphoid cells or myeloid cells.

Those destined to the lymphoid path start out as lymphoblasts and will further develop into different types: B-lymphocytes, T-lymphocyte, or plasma cells. Those destined to the myeloid pathway also start out as blasts and will further develop into one of the other four types of white blood cells (neutrophils, monocytes, eosinophils, or basophils), red blood cells, or platelets.

When examining bone marrow cells prior to their specialization towards a specific type of cell (i.e. the “blast” cells), they are virtually indistinguishable from each other based on appearance alone. There are no accurate ways to simply look at a very primitive blast cell and know whether it is destined to become a lymphocyte, a neutrophil, or a monocyte.

In leukemia, somewhere along the maturation process within the bone marrow, a single cell begins dividing uncontrollably and the cells are released into the blood stream where they can cause the total white blood cell count to rise and also accumulate within lymph nodes, where they can then cause these organs to enlarge. The tricky part is the same changes (abnormal cells in circulation and enlarged lymph nodes) are seen with pets with lymphoma as well.

These abnormal cells are often picked up on routine blood tests or can be noticed via an aspirate of an enlarged lymph node. The abnormal results are usually noticed and a lab technician or clinical pathologist will evaluate a blood smear and validate results.

153 Testing Bone marrow cytology is a test considered part of routine staging for pets with any hematological (blood-borne) cancer. Bone marrow analysis provides information as to what percent of this tissue is comprised of cancerous blast cells, which is useful in distinguishing lymphoma from acute leukemia. Most dogs with lymphoma have a low- level of cancer cells in their bone marrow, however if the percent of blast cells exceeds >20-30 percent of the entire sample, it is more typical for a case of leukemia.

Bone marrow cytology can be inaccurate in determining the exact cell of origin of the abnormal cells in question.

Flow cytometry is designed to look for specific markers located on the surface of cancer cells to aid in determining their origin (e.g., whether lymphoid or non-lymphoid in origin). This test can be performed on blood, bone marrow, and also fine needle aspirates of tissues (e.g., lymph nodes). One of the main markers this test can examine for is called CD34. In general, cells of bone marrow origin will express CD34, whereas those located in the periphery of the body will not. If detected, the presence of CD34 strongly supports the diagnosis of an acute leukemia.

PCR for antigen receptor rearrangement (PARR) is a DNA based test. PARR is only valuable for testing lymphocytes, so when we choose this test, we must be at least reasonably certain the cells in question in our samples are lymphocytes. Additionally, PARR cannot distinguish lymphoma from acute leukemia of lymphocyte origin. Essentially, what PARR tells us is 1) If the sample is from a cancerous condition of lymphocytes, and 2) if it is of a B-lymphocyte or a T-lymphocyte origin.

Cytochemistry staining is similar to flow cytometry. This type of test looks for markers on the surface of, or within, white blood cells. An equivalent of this test on a biopsy sample would be called immunohistochemistry.

Treatment Lymphoma treatment uses chemotherapy as the most common treatment. It can be administered orally or intravenously. It is common to have hair loss, experience vomiting and diarrhea, and have low blood cell counts. Because chemotherapy causes immunosuppression, there is an increased risk for infection. Radiation treatments may be recommended instead of or in combination with chemotherapy as a more targeted treatment. Whole or half-body radiation is used since the cancer is not usually contained to one area. Surgery may also be an option for localized lymphoma that only affects a single lymph node. Usually this has the best chance for treatment success since it is more likely that the lymphoma has been caught early on before metastasis.

Acute leukemia can be treated with aggressive chemotherapy, while continued monitoring may be recommended for chronic leukemia.

154 TECHNIQUES FOR THE DIAGNOSIS AND CLASSIFICATION OF LYMPHOMA AND LEUKEMIA Dr. Anne Avery, VMD, PhD

A variety of complementary modalities are used for the diagnosis and classification of lymphoma and leukemia. Cytol- ogy and/or histology are frequently the first steps, but these methods alone often do not provide prognostic informa- tion or point to clear treatment choices. Clonality testing can be used to establish if a malignancy is present when other diagnostics are ambiguous. Flow cytometry is now commonly used to subclassify these diseases into clinically relevant groups with different outcomes and treatment choices. Flow cytometry is also used to measure the prolifera- tion of neoplastic cells, which provides prognostic information in some tumors, such as B cell chronic lymphocytic leukemia. Other methods, such as gene expression and mutation analysis, are just on the horizon and will almost cer- tainly be in common use in the next 3 – 5 years. The goal of this session will be to describe what testing methods are most useful for achieving a diagnosis and prognosis, the principles underlying those methods, and the technical aspects of collecting samples.

155 CRYOSURGERY: A SUPER COOL TREATMENT OPTION Aspen Schreiner, CVT

The use of cold temperatures in medicine has been around for thousands of years, dating back to the ancient Egyptians. Although initially used to alleviate pain and inflammation, the use of “cold” has been extended to cancer therapy (specifically used to cause severe tissue damage). Cryotherapy refers to the therapeutic use of cold against pain and inflammation, while cryosurgery is defined as the “controlled use of substances that produce freezing temperatures cold enough to damage tissue severely”. Cryogens are the substances used to create the freezing temperatures that cause tissue damage. Examples of cryogens include liquid nitrogen and liquid nitrogen substitutes.

The freezing of tissue begins at 0º C with the crystallization of water in the spaces between cells. Freezing tissue can be achieved either through a slow or rapid freeze, with thawing of tissue following suit. Cryosurgery utilizes both the rate of freeze as well as the rate of thaw. The optimal combination to treat malignant tumors being rapid freeze, slow thaw, followed by consecutive freeze-thaw cycles.

Cryogens may be applied by use of a probe, or by pouring the liquid directly on the targeted area. Devices such as cryocones should be used to ensure the cryogen stays within the treatment area.

Cryosurgery is ideal for small tumors of the skin, oral cavity, and anus. Advantages include lack of hemorrhage, lack of systemic effects from freezing, and ease of repetition. Disadvantages include sloughing of skin, scar formation, as well as weakened bone, and rapid degranulation of mast cell tumors.

156 ELECTROCHEMOTHERAPY Brooke Quesnell, B.S, CVT, VTS (Oncology)

INTRODUCTION: Electrochemotherapy, a local treatment of various neoplasms, utilizes electroporation to change the permeability of cell membranes to facilitate the uptake of cytotoxic drugs. These drugs, injected directly into the tissue, would otherwise be unable to pass through the cell membrane efficiently. Bleomycin and cisplatin are the two drugs shown to be effective with this treatment modality, although other drugs are being researched. Electrochemo- therapy’s mechanisms of action include: increased membrane permeability and intracellular drug accumulation; vas- cular effects; and involvement of immune response.1 Electrochemotherapy has been shown to be an effective treatment for a variety of neoplastic processes and has been shown to be completely effective in as little as 1 treatment. It is best utilized to treat superficial tumors or areas where surgical excision was incomplete or unable to be performed at all. Side effects are typically limited to local tissue inflammation and necrosis. Performing electrochemotherapy requires heavy sedation or, ideally, general anesthesia. Proper safety protocols must be used to protect not only the patient, but all staff involved to reduce risk of exposure. Overall, electrochemotherapy can be an effective treatment for various malignancies with minimal side effects.

ELECTROPORATION: Electroporation is the process of introducing a substance into a cell using a pulse of electric- ity to temporarily open the pores in the cell membranes. Application of electrical current to the cells causes relocation of the charges on the cell membrane. This relocation of charges alters the cells membrane potential allowing the forma- tion of pores through which water, charged molecules, and larger molecules may pass into the cell. Various levels of electrical pulses have been studied and the standard dose of 8, 1,000-1,500V/cm, 100-μs-long pulses.1,3 This process is what makes electrochemotherapy successful. By allowing the drug to directly enter the cell, intracellular drug concen- trations are 2-4 times higher than traditional IV administration techniques.3

Vascular Lock: Vascular lock is the term used to encompass two vascular effects caused by the electrical pulses applied in electrochemotherapy. Applied electrical fields assist stromal cells with drug uptake and effect endothelial cells of the tumor’s vessels. This effect, termed vascular disrupting effect of electrochemotherapy, leads to endothelial cell apoptosis and therefore disruption of tumor blood flow. The second effect is vasoconstriction in the area the electrical field is ap- plied. These two effects combined “lock” the administered drug within the applied electrical field area creating further exposure to the desired tissue. If a chemotherapeutic is administered after the electrical pulses have been applied, it can block the drug from entering the tumor.1

Immune response: Studies have shown a difference in electrochemotherapy’s effectiveness between immunocompetent and immunocompromised patients. This difference implied an immune response is key to electrochemotherapy being a successful treatment. After electrochemotherapy, tumors shed immense amounts of tumor antigen which can induce a systemic immune response. This immune response can be up-regulated by biological response modifiers like IL-2, GM-CSF and TNF-α.3

BLEOMYCIN: Bleomycin is an antitumor antibiotic. It exerts its action by inducing DNA strand breaks, although the exact mechanism of action is yet to be fully understood. Bleomycin is most commonly used in electrochemo- therapy as it has a higher potentiation of cytotoxicity when an electrical pulse is applied. Bleomycin can be given intratumorally and intravenously during an electrochemotherapy procedure. Dosing parameters range from clinician to clinician but a sufficient maximum dose of 15U/m2 per treatment is commonly utilized. Dilution of the bleomycin should be done according to manufacturer specifications. Depending on the size of the area being treated, most of this dose may be given intratumorally with the remainder being administered IV. The IV administration should be per- formed first, then the intratumoral(IT) injection due to the vascular lock effect. The IV portion of the drug, if appli- cable, should be given 4-8 minutes prior to the IT injection. Electrical pulses should be applied within 1 minute of the IT injections.

CISPLATIN: Cisplatin is the second most commonly used drug in relation to electrochemotherapy. Cisplatin, a plati- num-agent chemotherapeutic, binds to DNA and interferes with replication. Cisplatin is contraindicated in cats due to

157 severe renal toxicity and typically fatal pulmonary toxicity. Studies have been done however using electrochemotherapy with cisplatin in cats that have had mild toxicity concerns and positive treatment outcomes.4 Dosing parameters for cisplatin also vary between clinicians but a sufficient maximum dose per treatment is 15mg/m2. In dogs, the cisplatin dose may also be given IV and/or IT, with IV administration occurring first.

MALIGNANCIES: A variety of malignancies have been proven to be sensitive the electrochemotherapy treatment on both canines and felines. Squamous cell carcinoma, fibrosarcoma, localized lymphoma, mammary adenocarcinoma, anal sac carcinoma, apocrine gland carcinoma, mast cell tumors, melanoma, and perianal tumors have all shown prom- ising responses to electrochemotherapy treatment. A meta-analysis of 1,894 tumors from 44 eligible clinical studies published before October 2011 demonstrated that the effectiveness of single-session electrochemotherapy on cutane- ous and subcutaneous tumors is 59.4% for complete response and 84.1% for objective response.1

PROCEDURE: Electrochemotherapy is a relatively quick, inexpensive treatment for a variety of tumor types. For the procedure patients should be under general anesthesia. Deep sedation can be used as an alternative but is less ideal. Anesthesia induction and maintenance protocols may vary due to clinician preference. The patient should be posi- tioned as necessary on a table that allows easy access for the anesthetist as well as the clinician and their technician. The clinician will be responsible for the IT injections as well as the application of the electrodes. The technician assisting will be responsible for containing any rogue chemotherapeutic agent as well as operating the electroporator as instruct- ed by the clinician. Isolation of the area being treated is necessary to prevent unnecessary drug exposure.

The area being treated should be clean and free of debris. The chemotherapeutic of choice should be injected into the area of treatment to sufficiently encompass all potentially malignant tissue. Recall that any portion of the drug to be given IV should be done so before the IT injections. Once the chemotherapeutic has been adequately administered into the tissue, electrical pulses should be applied within 1 minute. For the use of plate electrodes, a water-based gel should be applied to the tissue in question before applying an electrical pulse. The application of the electrode should start from the safety margin of the tumor and progress towards the center in a circular motion. Applying the electrode twice in a perpendicular pattern will ensure the most complete application of electrical field. Gel application is not necessary when using a needle electrode. All needles should be inserted into the tissue to ensure proper electrical field application. The pattern described above for the plate electrode also applies to the needle electrode. The application of electrical pulses will cause a muscle contraction in the patient. Depending on the area of pulse application measures should be taken to restrain areas of the patient’s body to protect the patient and those around it.2

SAFETY: As with all chemotherapy administration techniques, extreme care must be taken to protect all persons involved in the procedure from exposure to the drug in question. Full personal protective equipment must be utilized. This includes a non-permeable gown, chemotherapy approved gloves, eye protection (preferably goggles) and at least an N-95 filter mask. The procedure should take place in a dedicated chemotherapy administration suite as contamina- tion risks are high. Ideally the suite should have negative air pressure to prevent flow of contaminated air into other parts of the building. Chemotherapeutic agents should be prepared in a class II laminar flow biologic safety cabinet to collect any aerosolized particles that may be produced during preparation.

The area being treated should be isolated as much as possible using non-permeable chemotherapy pads, gauze, or other absorbent material to collect any leaking chemotherapeutic. Care should be taken on the part of the clinician to limit unnecessary injection, leakage, or spray of the chemotherapeutic agent. All disposable materials utilized during the procedure should be discarded in an appropriate chemotherapy waste or sharps container. Tools utilized such as the electroporator, anesthetic machine, and monitoring equipment should be thoroughly wiped down with Lysol wipes or with paper towels that have been sprayed with a Lysol or bleach solution. Tools or contaminated surfaces should not be sprayed directly as this could potentially cause aerosolization of chemotherapeutic agents. The area treated on the patient should be cleaned thoroughly post-electrochemotherapy to ensure no residual contamination is present.

Should a person be exposed to a chemotherapeutic agent the area should be flushed copiously with water. Should a contaminated needle stick occur, flush the area copiously with water as previously advised and seek medical attention

158 as needed. Bleomycin and cisplatin rarely cause acute effects with skin surface contamination, although irritation may occur.2 Every safety measure should be utilized to prevent any exposure to these agents as long-term exposure has been linked to a variety of significant medical issues.

CONCLUSION: Electrochemotherapy is a promising treatment for a variety of superficial malignancies. The proce- dure is affordable, fast, and less invasive when compared to surgery and/or radiation. Appropriate care must be taken to perform the procedure properly and to protect all those involved from exposure to chemotherapeutic agents. Fur- ther research and studies will likely lead to further applications of electrochemotherapy for cancer treatment.

REFERENCES 1. Miklavcic D, et al. Biomed. Eng. Online 2014;13:29 2. Tozon N, et al. J. Vis. Exp. 2016; 116 3. Sersa G, et al. Radiol Oncol 2006; 40(3):163 4. Spugnini EP, et al. J Transl Med 2011; 9:152

159 CHEMOTHERAPY SAFETY AND USP800 COMPLIANCE “FAQ’S GALORE” An Open Dialogue Jenny Fisher, RVT, VTS-Oncology

The National Institute for Occupational Safety and Health (NIOSH) have set criteria for what “Makes a HD”. The six criteria include: carcinogenicity, teratogenicity, reproductive toxicity, organ toxicity at low doses, genotoxicity, struc- ture, and toxicity profiles that mimic drugs deemed HD by previous stated criteria. These criteria were originally set in the late 1970’s – years before genetically modified drugs and some of the other treatment modalities available today. As many older regulations, these also need to be adjusted based with progressive modern medicine. Many groups/govern- ing bodies exist to evaluate and develop safety protocols based upon specific criteria. One of these agencies is USP. The most current hazardous drug handling recommendations are outlined in USP 800.

Implementing these new guidelines will require hospitals to adapt new technology, gain additional training and potentially change the facility physical structure. Four main points of USP 800 require the use of PPE, CSTD, class IIb hood and a negative pressure room. All four of these points will be required for any facility to pass compliance. Enforcement will mainly lie within state regulations and it is unclear at this time the true impact financially. Routes of potential exposure are also included in 800, including receipt, unpacking and storage of HD.

Chemotherapy safety is a constant topic for veterinary oncology personnel. Over the past few years, with the publish- ing of USP800 many veterinary facilities have been trying to understand the recommendations and become compli- ant. Safety guidelines and topics are typically not a subjective conversation. This lecture will cover updates and fre- quently asked questions regarding USP800 compliance.

160 IMPLEMENTING STATE-OF-THE-ART RADIATION THERAPY IN VETERINARY PRACTICE Dr. Kelsey Ericksen Pohlmann, DVM, DACVR-RO

• Outline: o Case report – Lexi o What is radiation? o Physics o Biology o The evolution of radiation therapy o Indications for referral o Implementing SRS in veterinary practice o Where YOU come in • What is Radiation? o Electromagnetic energy o Light waves, radiowaves, microwaves… o X-Rays! • Types of radiation o Electromagnetic . X-Rays ◊ Produced in an x-ray machine or linear accelerator ◊ This will be the focus of our discussion . Y-Rays ◊ Emitted from radioactive elements ◊ Cobalt units ◊ I131 . Particulate ◊ Electors, Protons, Heavy charged particles • X-Rays o Electromagnetic energy . Encountered everyday . The x-rays pass through the subject ◊ Absorbed – Biological effects in tissue ◊ Transmitted – Captured by film or scattered . Patients are not radioactive . Radiation is not excreted in any form after the imaging or treatment is performed o Diagnostic: . Low energy (kV) . Differential absorption in tissue . Bone absorbs more than soft tissue, fat, lung . Film absorbs the x-rays that were not absorbed by the patient – shades of gray seen on film o Therapeutic . High energy (MV) . Absorbed evenly across all tissues . Biologic effects in tissue/cells – DNA damage • What is Radiation Therapy? The Biology: o X-rays damage DNA of cells o Cells die when attempting to replicate/multiply o Interference with replication – grow inhibition o Non-invasive, pain-free therapy o Localized treatment • Response to DNA damage o Repair . Many mechanisms . “Normal” cells have intact DNA repair mechanisms – repair is more likely in healthy tissue o Cell death . Cancer cells have faulty DNA repair mechanisms

161 . Cancer cells are more likely to die when DNA is damaged . Mitotic death (mitosis = cell division) ◊ Cell dies when attempting to copy its DNA and divide . Apoptotic cell death ◊ Occurs in particular cell types o Lymphocytes o Vascular endothelial cells • Tumor response and cell kinetics o Kinetics = rate of a reaction . Here, we are referring to what happens to a tumor after radiation as an implication of the tumor’s cellular kinetics (rate of growth) . Tumor cells die when they are trying to divide/multiply ◊ Rapidly growing tumors should die/shrink rapidly ◊ Slowly growing tumors should die/shrink slowly/gradually . What owners need to know: ◊ Most tumors will not shrink overnight ◊ Prepare for a marathon, not a sprint • Normal tissue response and cell kinetics o Same is true for normal tissues! . Tissues that turnover quickly à show side effects early/shortly after treatment ◊ Acute radiation effects . Tissue that turnover slowly à show side effects months/years after treatment ◊ Late radiation effects o Acutely responding tissues . Rapid rate of cell turnover . Show side effects days-weeks after treatment . Recovery time is usually quick and complete . Skin, mucosa, GI tract, hematopoietic system o Late responding tissues . Slow rate of cell turnover . Show side effects months-years after treatment . Recovery, if it occurs, is slow and often not complete · Late radiation effects are dose limiting . CNS (brain, spinal cord), bone, lung, kidney, lens • Acute radiation effects o Timeline: . Time from radiation – side effect = time for a stem cell to become a mature/functional cell . What? ◊ The surface of your skin will slough tomorrow – damage here is not important ◊ The stem cell layer of your skin will “rise” to the surface in 10-14 days – damage here is important! ◊ And the damage will show up when those stem cells reach the surface = 10 – 14 days post-treatment for skin • Acute radiation effects o Timeline: . Time from radiation – side effect = time for a stem cell to become a mature/functional cell ◊ Skin = 10-14 days ◊ Mucosa = 7-10 days ◊ GI = 5-7 days ◊ Timeline for recovery = similar • Late radiation effects o Pathogenesis less clear than acute effects . Fibrosis, necrosis, vascular changes . “Failed” healing

162 o Generally considered irreversible - dose limiting . 5% risk considered acceptable . Incidence with SRS comparable to CFRT/IMRT ◊ Severity may be worse ◊ “5% feels like 100% if it happens to your pet • Radiation Therapy – The Evolution o Clinical Setup/”Point-and-Shoot” Radiation Therapy o Intensity Modulated Radiation Therapy (IMRT) o Stereotactic Radiosurgery (SRS, SRT, SBRT, SABR) o Volumetric Modulated Arc Therapy (VMAT, RapidArc®) • Radiation Therapy – CFRT o Conventionally Fractionated Radiation Therapy (CFRT) o Widely available o 15-21 treatments (“fractions”) and anesthetic events o Standard of care for incompletely excised tumors . Mast cell tumors, soft tissue sarcomas, AGASACA o Healthy tissue will repair over time, but dose per fraction must remain low to avoid severe toxicity = spare normal tissues by fractionation • Targeting tumors: Multi-leaf collimator – Static o Allows better conformity to decrease normal tissue damage • Radiation Therapy – IMRT o Intensity Modulated Radiation Therapy (IMRT) o Dynamic multi-leaf collimator à non-uniform fluence o Multiple beams à steep dose gradient o Inverse treatment planning . Patient-specific, CT-based identification of tumor target and organs at risk o Requires accurate, precise and highly reproducible patient positioning o 15-20 fractions • What is Stereotactic Radiosurgery (SRS)? o More Precision o We can treat tumors in locations previously thought “untreatable” . Lung, liver, kidney, prostate, adrenal gland, heart base, etc… o Fewer Treatments . Treatments given in 1-3 consecutive days (instead of 15-20) . Fewer anesthetic events and trips to the hospital o Fewer Side Effects . Radiation side effects happen when healthy tissue is exposed to radiation – more precision means those side effects are greatly reduced • Indications for Referral • Cancer Treatment Options o Chemotherapy o Surgery o Radiation o Immunotherapy o Palliative care • Radiation Therapy o Local therapy o Consider when surgery not feasible or is unlikely to achieve clean margins o Can result in side effects to healthy tissues • Indications for referral: o When there is residual microscopic disease . Post-surgery dirty margins (mast cell tumor, soft tissue sarcoma) . Conventionally fractionated radiation is standard of care to minimize risk for tumor regrowth

163 o Bulky disease, where a good surgical option does not exist . Location . Ability to achieve clean margins . High morbidity o Advanced stage disease . Palliative radiation addresses local/clinical disease • Implementing SRS in Veterinary Practice • PetCure Preliminary Data o Patients with incomplete datasets not included in this analysis o Censored = . Alive . Lost to follow-up . Death from unrelated cause o All analysis NCSS v11.0 o Study power suboptimal in many cases • Tumors commonly treated with radiation therapy – Canine Nasal Tumors o Current literature: . MST no treatment = 95 days . MST surgery = 3-6 months . MST CFRT/IMRT = 11-19.7 months ◊ Stage 4 (cribriform lysis) = 6.7 months . MST SRS literature = 12-20 months o PetCure Median Survival Estimates: . Carcinoma = 634 days (132 cases, 80 censored) ◊ 21 months . Chondrosarcoma = 710 days (19 cases, 15 censored) ◊ 23.6 months . Squamous cell carcinoma = 273 days (17 cases, 7 censored) ◊ 9 months • Tumors commonly treated with radiation therapy – CNS* • Canine brain tumors – presumed meningioma . MST no treatment = 2-3 months . MST surgery + CFRT = 11-19 months . SRS literature = 18 months (Griffin, CSU 2014) ◊ Meningiomas only o PetCure Median Survival Estimates: . Glioma = 622 days (30 cases, 20 censored) ◊ 20.7 months ◊ Previously reported with CFRT = 8-10 months . Meningioma = 647 days (117 cases, 85 censored) ◊ 21.5 months . Median survival estimate = 20.7 months . Previously reported with CFRT = 8-10 months • Tumors commonly treated with radiation therapy – Osteosarcoma o Canine OSA . MST palliative care only (pain meds) = 1-3 months . MST amputation alone = 3-6 months . MST amputation + chemotherapy = ~1 year (8-12 months) . SRS experience = ◊ Bisphosphonate therapy prior to treatment ◊ Chemotherapy indicated following SRS ◊ MST = ~12-15 months • Tumors commonly treated with radiation therapy – Oral Tumors o Oral tumors . Prognosis dependent on histotype . Local control generally good (>12-18 months_ . Call before performing dental procedures!

164 • Tumors commonly treated with radiation therapy – Canine soft tissue sarcoma* o SRS . 43 total cases, 38 censored . 19-1624 days . 75% Quantile estimate: 944 days (31 months) o All had gross disease o No systemic therapy in this group o Tumors confined to the skin/subO • Tumors commonly treated with radiation therapy – Canine soft tissue sarcoma – Clinical Trial* o Nanovi PetXMark™ o Marginally resected grade II sarcoma with a linear scar . <15cm . Liquid fiducial used to define a surgical scar . Other tumors of the skin/subO can be treated off protocol . PetCure will retreat in the event of early local failure or geographic miss · This clinical trial is closed for ongoing enrollment • Nanovi Trial: Results, All Tumors o 173 patients injected to date . 161 treated ◊ 12 rejected for comorbidities . 144 alive . 11 Dead not tumor . 5 dead to tumor . 1 lost to follow o 1 possible/presumed reaction to the injection o 2 confirmed local recurrence o 2 significant local toxicity cases . 1 owner non-compliance with prevention of self-trauma . 1 developed fungal infection after swimming in a lake o Standard of Care = CFRT . 18-20 daily treatments . Moderate-severe acute toxicity that lasts 4-6 weeks . 85% chance for long-term local tumor control • Tumors commonly treated with radiation therapy o Lung . Alternative to surgery for primary lung tumors . SRS experience – local control >12 months . Human experience: SRS achieves similar or better outcome compared to surgery as primary treatment for non-small cell lung cancer . Currently enrolling canine patients for prospective clinical trial of SRS for primary lung tumors • Canine Lung Tumor Clinical Trial o Solitary lung tumor o No life threatening comorbidities o SOC1 = 3x20Gy o SOC2 = 1x24Gy o Trial Closed July 2020 • Tumors commonly treated with radiation therapy – Thyroid tumors * o Descriptive data . 23 total cases, 16 censored . 18-808 days o Median survival estimate 627 days = 21 months o Most of these cases have massive tumor volume and were deemed inoperable • Tumors commonly treated with radiation therapy – Adrenal tumors* o Descriptive data . 5 total cases, 5 censored . 4-632 days o No survival estimates possible at this time – no uncensored cases

165 • Side Effects with SRS o Acute toxicity is significantly reduced with SRS, but patients may still experience side effects in the location of the treatment. If we never saw side effects, it would mean we weren’t treating the cancer aggressively enough. • Side Effects of Radiation Therapy o Acute (early) effects . Days-Weeks after treatment ◊ Correlates with short life span of mature cell population . Rapidly dividing tissues ◊ Skin, mucosa (oral, GI), bone marrow . Moderate-severe with CFRT . Mild-Moderate with SRS . None-Mild with palliative radiation . Should resolve without intervention, though supportive care may be required o Treat symptomatically! . Will resolve with time, prevention of self-trauma and appropriate supportive care o Skin desquamation: . ~10-14 days post-SRS; resolves in ~10-14 days . Pain medication . Prevention of self-trauma . Keep clean/dry ◊ But don’t encourage owners to clean/groom/etc ◊ Do not bandage . Antibiotics for secondary infections o Skin . Hair loss . Permanent hair coat color change . Hyperpigmentation . Mild-moderate inflammation/desquamation o Treat symptomatically! . Will resolve with time, prevention of self-trauma and appropriate supportive care o Mucositis . ~10-14 days post-SRS; resolves in ~7-10 days . Anti-inflammatory dose of steroids . Magic mouthwash à compounding pharmacies . – +/- pain medication as needed . Consider softening food or providing canned food . Avoid hard chew toys o Colitis . ~5-7 days post-SRS; resolves in ~3-5 days . Metronidazole à owners are sent home with this if anticipated o Ocular changes à refer to ophthalmologist . Dry eye . Conjunctivitis o Early-delayed/transient demyelination . 3-6 months post-SRS ◊ Reported in 20-40% of patients . Recurrence of initial clinical signs . Steroid-responsive . Treatment = aggressive supportive care and steroid therapy . SLOW steroid taper following recovery ◊ 25% every 4 weeks if no relapse o Late effects . Months-years after treatment . Slowly proliferating tissues ◊ Brain, spinal cord, heart, liver, lung, bone . May improve, but never completely repaired

166 o Goal = minimize risk . 5% risk acceptable • Where do YOU come in? o The role of veterinary technicians in radiation oncology • Role of Veterinary Technician in Radiation Oncology o Patient evaluation . Record review . Medical history . Triage . Diagnostics ◊ Bloodwork ◊ X-rays o Explanation of process . Reviewing estimates . “Show and tell” . Treatment plan review . Discussion of anticipated side effects and follow up recommendations o CT Simulation . Anesthesia ◊ Induction ◊ Monitoring ◊ Recovery . Determine ideal positioning . Create immobilization devices . Acquire images o Treatment . Anesthesia ◊ Induction ◊ Monitoring ◊ Recovery . Reproduce position . Image verification . Beam on! • Role of the Veterinary Technician in Radiation Oncology – Invaluable Skill o General oncology knowledge o Strong anesthesia skills o Strong background in anatomy o Experience reviewing CTs o Attention to detail

167 DIVING INTO DIAGNOSTIC IMAGING Madeline Ederer, RVT, VTS (Oncology)

Diagnostic imaging is an integral part of veterinary oncology. It is used to help diagnose, plan treatments, assist with tissue sampling, monitor disease progression, and more. This presentation will explore the various imaging modalities frequently used for oncology patients in veterinary medicine. Examples and techniques of common imaging such as radiographs, as well as advanced imaging such as MRI, will be provided. A brief review of how anatomy should appear in each type of imaging will enable faster interpretation of the images, ultimately leading to better patient care.

I. Overview

II. Importance in Veterinary oncology a. Diagnosing b. Staging c. Treatment planning d. Monitoring

III. Modalities a. Common imaging i. Radiography ii. Ultrasound b. Advanced imaging i. Computed tomography (CT) ii. Magnetic resonance imaging (MRI) iii. Positron emission tomography (PET) iv. Nuclear scintigraphy

IV. Techniques relevant to veterinary oncology a. Metastases b. Bone lesions c. Image guided diagnostic testing

V. Normal vs. neoplasia a. Skull, thorax, limbs, abdomen, pelvis

VI. Conclusion

168 Case Report

GIVING UP THE COMMITMENT TO THE PROBABLE: DURABLE REMISSION IN A CASE OF FELINE LARGE CELL GI LYMPHOMA Rachael Rail, RVT

Introduction: Lymphoma is generally defined as a cancer of lymphocytes, which are a type of white blood cell that is present throughout the entire body. Lymphoma usually arises from lymphoid tissues such as the spleen, lymph nodes and bone marrow, but can arise from almost any tissue in the body. It is one of the most common forms of cancer in felines and is the most common tumor type found in the feline gastrointestinal (GI) tract.

Feline GI lymphoma can be categorized as one of three types based on histopathology and immunohistopathology findings. The three types of feline GI lymphoma include low-grade alimentary (LGAL), intermediate or high-grade alimentary (I/HGAL), and large granular lymphoma (LGL).

Felines with large cell GI lymphoma (HGAL) often present with non-specific GI signs and weight loss that have acutely progressed. They are more likely to present with a palpable abdominal mass, enlarged mesenteric lymph nodes or liver than those with LGAL. Icterus also more commonly coincides with large cell GI lymphomas. They are often diagnosed via abdominal imaging and cytology from an abdominal/mesenteric lymph node or lesion found in the GI tract.

The prognosis for felines with HGAL is, unfortunately, poor. The disease is very aggressive and is often difficult to treat. Even with aggressive chemotherapy, only about 30% of felines respond and typically have an average survival time of two to three months. There is a very small percentage of felines who completely respond to chemotherapy and can survive for close to a year.

Case History: Guido, a 15-year-old neutered male Bengal feline was presented to the ISU Emergency service for progressive lethargy, anorexia, and weight loss. He was 5% dehydrated and anemic with a hematocrit of 24% on presentation. An abdomi- nal ultrasound revealed a 3cm jejunal thickening and multiple enlarged mesenteric lymph nodes. Due to the vascular nature of the intestinal lesion, fine needle aspirates were taken of the regional lymph nodes. The results were suggestive of large cell lymphoma.

Treatment Methods: Guido received a blood transfusion due to his anemia. The purpose of the transfusion was to better perfuse and oxygenate his tissues, which would hopefully improve his clinical condition. His anemia was most likely due to blood loss via the intestinal wall mass found on abdominal ultrasound.

An esophageal feeding tube was placed under general anesthesia. This was recommended due to his hyporexia, and to offset the break-down of muscle and fat at a higher than normal rate that occurs when a patient has cancer. His feeding tube remained in place for the entirety of his treatment and remission and allowed his owner to be actively involved in his care. She was able to medicate, hydrate and feed him via the esophagostomy tube when necessary.

169 Surgical excision of the intestinal lesion was discussed to achieve a definitive diagnosis. Although his risk of GI perfora- tion was significant, surgical removal of the lesion was declined.

Guido was started on a CHOP chemotherapy protocol. He was treated weekly for 3 weeks with Vincristine (week 1), Cyclophosphamide (week 2), and Doxorubicin (week 3) followed by a one week break. This cycle was completed 6 times for a total of 24 weeks of treatment and appropriate treatment delays/substitutions as needed for expected toxicity. Guido was switched from Doxorubicin to Mitoxantrone during the course of his treatment due to progressive kidney disease. In felines, Doxorubicin can cause a cumulative nephrotoxicity, unlike canines, who can experience cardiotoxicity, and should be used with caution when feline patients have underlying renal disease. Mitoxantrone is much less nephrotoxic to felines and can be a reasonable alternative when Doxorubicin is contraindicated.

An abdominal ultrasound was performed on the day of his last chemotherapy treatment and he was found to be in remission at that time. After receiving his final dose of Mitoxantrone, a long term recheck plan was made and Guido continued to receive recheck abdominal ultrasounds every month to look for evidence of disease progression. He also received routine blood work evaluations to monitor his anemia and kidney disease. During his treatment and remission, he received multiple blood transfusions due to progressive non-regenerative anemia of undetermined etiology after extensive diagnostics.

Results and Outcomes: Guido’s remission lasted for 17 months after finishing his full CHOP chemotherapy protocol. He defied the odds and outlived even the smallest percentage of felines by five months. During the course of his treatment and remission, Guido battled multiple co-morbidities that we, as his medical team were challenged to balance. These co-morbidities included renal disease, glaucoma, cardiac disease, non-regenerative anemia, demodex, and seizures. He was humanely euthanized a year and a half after his initial diagnosis due to clinical decline and suspected progression of his lymphoma.

Moving Forward: Guido has been a very influential patient during my career in Oncology. Moving forward, I will always be an advocate for esophagostomy tubes in my feline lymphoma patients. As we all know, felines can be tricky to treat and medicate. I truly learned the value of a feeding tube with Guido and I feel that it should be standard of care for our feline patients going through chemotherapy. It not only makes it easier to provide them with essential nutrients during chemotherapy, but it increases their quality of life and allows their owners to take an active role in their treatment.

In addition, I would also advocate for venous access port placement in felines receiving multiple rounds of chemo- therapy. Guido did not have a port during his treatment and it was the most stressful part of his care for both of us. His veins were unforgiving and there were days when IV catheters were not able to be placed safely and treatment was delayed. Often times, the prognosis for felines with lymphoma is poor, so placing a port is not seen as the most cost- effective procedure when weighing it against expected survival times.

References: Vail, D. M., Thamm, D., & Liptak, J. (2019). Withrow and MacEwen’s Small Animal Clinical Oncology - E-Book (6th ed.). Elsevier Health Sciences. O’Keefe DA, Sisson DD, Gelberg HB, et al.: Systemic toxicity associated with doxorubicin administration in cats, J Vet Intern Med 7:309-317, 1993.

170 CANCER GRADING & STAGING: UNDERSTANDING THE BASICS Dr. Zachary Neumann, DVM, MS, DACVIM (Oncology)

A basic understanding of grading and staging is necessary as an oncology technician. How the oncologist approaches cancer patients in general terms provides the foundation to understanding individual cancer types. With technicians having heavy involvement in communicating with clients, they need to understand how to describe the terminology around cancer care with confidence. Browsing previous proceedings from VCS, I do not see any previous sessions dis- cussing in depth the diagnostic approach as a whole; rather, most sessions have focused on individual diagnostics. This interactive presentation will focus on tying together all the diagnostics in a pragmatic approach to help technicians understand why we perform the tests we do every day. We will start with a focus on the two most important questions to the oncologist: “What is it?” and “Where is it?”. We will then dissect the tests necessary to determine the answers to these two questions.

171 ONCOLOGY EMERGENCIES Dr. Andi Flory, DVM, DACVIM (Oncology)

Outline: • It IS the cancer! o Tumor-related emergencies; Treatment-related emergencies • It’s NOT the cancer! o Concurrent diseases; Non-cancer-related emergencies • Counseling oncology clients for emergency treatment Why should the oncology veterinary nurse learn about oncology emergencies? Oncology patients may present on emergency, for reasons related to their cancer, cancer treatment, or completely unrelated to the cancer treatment. Understanding the reasons that oncology patients may present on emergency and how those emergencies are treated is vital to educating clients on what to watch for, and when to bring their pet in to be seen. The oncology veterinary nurse’s role in client education cannot be understated. Vet nurses have a connec- tion with clients and are a great source of education for clients on what signs to watch for, what to do in case of severe treatment-related symptoms, when they should come in to the emergency service, and what they can expect if they come in for an emergency room visit.

Types of oncology emergencies Emergencies can be tumor-related (such as a bleeding tumor, painful tumor, or pathologic fracture), due to paraneo- plastic syndromes (such as a hypercalcemic patients with increased thirst and urination), or treatment-related (such as chemotherapy-induced febrile neutropenia or diarrhea, or radiation side effects). Non-cancer-related emergencies may be injuries (hit by car, bite wound), toxicities, or new/ progressive comorbidities (progressive kidney disease in older cats, heart failure) – sometimes these concurrent diseases may be made worse by the treatment, or by the cancer.

Tumor-related emergencies These are emergencies directly related to the cancer; patients may present to the emergency clinic with not-yet-diag- nosed cancer, or known but newly progressive cancer. This includes patients that might present for acute onset of “lumps under the chin” (dogs with lymphoma) that may be bouncing around and feeling great, and patients with a bleeding splenic tumor that are acutely anemic and have severe hemoabdomen and need emergency surgery. The job of the emergency clinician is typically to stabilize the patient, control symptoms, treat pain, and sometimes achieve a diagnosis so that cancer therapy can get underway. In some cases, such as lymphoma, the emergency clinician or criticalist at a specialty hospital might start cancer therapy (for example, give a dose of L-asparaginase) if all diagnostic testing is done, however in many cases, the pet will be stabilized to transfer to a specialist for treatment the next day.

Examples of types of tumor-related emergencies include: • Hemorrhage – bleeding peripheral tumors; epistaxis; bleeding into a body cavity (hemoabdomen, hemoperi- cardium, hemothorax); Yunnan Bai Yao is often advised • Body cavity effusions – neoplastic effusions in the thorax or abdomen; some patients may require therapeutic tap • Pain – ANY tumor can cause pain and all patients should be assessed for discomfort; if it’s ulcerated, bleeding, or bruised, it could be painful; NSAIDs, gabapentin, tramadol, Tylenol 3, fentanyl patch, etc Treatment-related emergencies Cancer therapies used in pets include surgery, radiation therapy, chemotherapy, immunotherapy, and a variety of other forms of palliative medical therapy, including corticosteroids, bisphosphonates, and pain medications. Chemotherapy for pets is generally well-tolerated, and adverse effects, if they occur, are typically mild and self-limiting. It is impor- tant for the oncology nurse to know the typical side effects that occur and the timing of those effects, to be able to properly counsel and educate clients regarding symptom management. • Radiation therapy effects include both acute and late toxicities, however the most common reason for emer- gency presentation would be acute. Moist desquamation, mucositis, colitis, rhinitis, otitis, conjunctivitis/ blepharitis might be common reasons for presentation, and depend on the site being irradiated. These effects often start the second week of radiation and may get worse after the end of radiation, before they heal. Com-

172 mon treatments include pain control, antibiotics, anti-inflammatories, and sometimes topical therapy. An E-collar is imperative to prevent self-trauma. • Chemotherapy side effects occur due to effects on rapidly-dividing cell populations. o The neutrophil nadir for most drugs is 7-10 days after chemotherapy, and typically neutrophil counts will rebound in about 36 hours. Patients that present with severe neutropenia (<1000) but normal body temperature are at high risk for infection and should receive oral antibiotics and discharged immediately. Patients that present with severe neutropenia and fever should be hospitalized for IV antibiotics and supportive care; these patients should also be worked up for a source of infection (e.g, pneumonia, skin infection, UTI). The prognosis is typically good for patients recognized early. o The platelet nadir for most drugs is 7-14 days, counts often return to normal within 1-2 weeks. Platelet counts do not typically go low enough to cause clinical symptoms of bleeding. • Immunotherapy or L-asparaginase could cause hypersensitivity reaction, such as hives, swelling, pruritus, or anaphylaxis. Non-cancer-related emergencies It’s important to remember that oncology patients can always develop other non-cancer-related medical conditions. They can ingest foreign bodies, get bitten by snakes, undergo trauma, or experience HGE just like any other pet. Cancer is most common in older patients, and older patients often have comorbidities. Heart disease, chronic kidney disease, diabetes, Cushing’s, IBD, chronic GI diseases like pancreatitis, arthritis, seizures might all be conditions that oncology patients have been previously or currently managed for, or they might be newly-diagnosed during cancer therapy. In some cases, the cancer or cancer therapy can cause comorbidities to worsen.

Client education and counseling • Clients should be educated on how to check their pet’s rectal temperature if receiving a myelosuppressive drug and instructed to check the temperature if their pet is not feeling well. The most common drugs that could cause neutropenia include lomustine, mitoxantrone, carboplatin, cyclophosphamide, and doxorubicin. A fever in a patient that has had chemotherapy in the past 2 weeks (or past 3 weeks for carboplatin) should be considered a medical emergency. • Pets should come in if: not acting like themselves, very lethargic, febrile, not eating or drinking for 12-24 hrs, diarrhea >3-6x over baseline, unable to take oral medications to treat symptoms, vomiting >3x or for >48 hrs, and/or not responding to medications. Err on the side of having them come in for evaluation! • Pet owners should be informed about the general routine of a visit to the ER, as it may be different than they are used to. An estimate for treatment is usually provided, and pets are often triaged in the treatment area, however the sickest and most urgent patients are treated first. The ER clinician will then examine the pet and provide consultation with the family on a recommended plan. Depending on whether the ER hospital is part of the same hospital system at which the pet is receiving cancer care, they may or may not have access to medical records, so clients should be instructed to bring medical records with them (often the most recent discharge instructions are best, as they will have an assessment and most recent therapy). In some cases, the emergency clinician will consult with the treating oncologist for help in managing the patient, but this is not always possible, or necessary. Preparing clients for emergency care is a key role of the veterinary oncology nurse, and can make the family more comfortable and the ER visit process go smoother.

173 MAST CELL TUMORS – WHAT YOUR MOTHER DIDN’T TELL YOU Dr. Pamela D. Jones, DVM, DACVIM (O), DACVR (RO)

I. Biology/incidence/behavior A. Up to 21% of canine cutaneous tumors B. Older dog – 7-9 years C. Breed predilection -Bulldog descent, Labradors, Staffordshire terriers, beagles, Rhodesian ridgebacks, Weimaraners, Shar-peis, others D. Cutaneous, subcutaneous, multiple E. Clinical signs 1. Variable appearance 2. Wax and wane 3. Degranulation – local and systemic

II. Diagnosis – background and controversies A. Fine needle aspirate and cytology B. Incisional biopsy C. Excisional biopsy

III. Grading – background and controversies A. Histopathology 1. Patnaik (1984) 2. Kiupel (2011) B. Cytologic 1. Scarpa (2014) 2. Hergt (2016) 3. Camus (2016) C. MCT prognostic panels D. c-KIT, mitotic index, Ki67, AgNOR

IV. Staging – background and controversies A. WHO Staging B. Lymph node (LN) assessment C. Abdominal ultrasound, organ aspiration D. Other 1. Thoracic radiographs 2. Bone marrow aspirate 3. Buffy coat analysis

174 V. Treatment – background and controversies A. Surgery 1. Local control 2. Surgical dose 3. Surgical margins vs histopathological margins (HTFM) B. Radiation therapy 1. Definitive fraction courses a) Adjunct to surgery b) Sole therapy 2. Stereotactic Body Radiotherapy 3. Palliative course C. Chemotherapy 1. Lomustine 2. Vinca-alkaloids a) Vinblastine b) Vincristine 3. Prednisolone 4. Combination a) Vinblastine/prednisolone b) Vinblastine/lomustine c) Vinblastine/lomustine/prednisolone D. Tyrosine kinase inhibitors 1. Toceranib phosphate (Palladia®) 2. Masitinib mesylate (Masivet in Europe) E. Other therapies 1. Electrochemotherapy 2. Intratumoral therapy a) Corticosteroids b) Others 3. Other local therapy

175 ANESTHESIA IN THE CANCER PATIENT Nicole Shuey BS, CVT, VTS (Anesthesia & Analgesia)

Anesthesia for cancer patients is more than just mass removals and biopsies. It poses unique challenges that the anesthe- tist must be prepared for including necessity of alterative pain management techniques, detrimental physiologic changes, and complex procedures to remove the cancer source.

Causes of pain Cancer pain can occur due to invasion of tumor cells into tissue, distension and obstruction of internal organs, or from inflammatory processes produced by cancer cells. Types of pain including somatic, visceral, and neuropathic pain. Each act more predominantly on different nerve fibers that are found in the body and create a different nociceptive response depending on the type of stimulus. Somatic pain is a type of nociceptive pain that is also referred to as skin pain, tissue pain, or muscle pain. Visceral pain stems from the activation of nociceptors of the thoracic, pelvic, or abdominal or- gans. Neuropathic pain arises from damage to the nervous system from trauma, infection, ischemia, systemic cancer, or chemically induced (chemotherapy). Some types of neuropathic pain may develop when the peripheral nervous system becomes damaged, causing the nociceptors to transmit pain signals repeatedly leading to hypersensitivity and peripheral sensitization. This abnormal input causes abnormal central processing and the persistence of hypersensitivity associated with neuropathic pain or chronic pain. Chronic pain persists beyond tissue healing, offers no useful biologic function or survival advantage, significantly affects a patient’s quality of life, and tends to be debilitating and poorly responsive to traditional analgesic therapy.

Pain not only causes patient suffering, which is stressful to both hospital staff and owners, it also affects our patients physiologically. It has been shown that pain produces a catabolic state (energy release), which may lead to muscle wast- ing. It also causes an increased anesthetic risk because higher doses of anesthetic drugs are required to maintain a stable plane of anesthesia. In addition, pain suppresses the immune response, predisposing the patient to infection or sepsis and increasing hospitalization time and cost. Pain also promotes inflammation which delays wound healing.

Pain management techniques Most patients diagnosed with cancer establish pain early in the disease, which intensifies rapidly as the disease progresses. The intent of pain management in these patients is to minimize peripheral and central sensitization and maintain quality of life. Pain management should therefore be multimodal and centered on chronic pain management. Analgesics medi- cations may include NSAIDs, opioids, NMDA antagonists, alpha-2 adrenergic agonists, anticonvulsants, and tricyclic antidepressants.

NSAIDs: Inflammatory pain, hyperalgesia, and allodynia (pain caused by stimulus that does not normally elicit pain) are often controlled with NSAIDs via COX inhibition. Some tumors overexpress COX-2, increasing prostaglandin genera- tion and inflammation. COX-2 inhibiting NSAIDs may also play an important role in management of these tumors. The most common side effects of NSAIDs include inhibition of platelet aggregation, impairment of renal perfusion, and inhibition of gastrointestinal protection.

Opioids: There is a wide range of narcotic medications that exert their effect on various opioid receptors (µ,δ , and κ). They can be administered IM or IV in the peri-operative period or orally for long term management. These drugs include hydromorphone, morphine, methadone, fentanyl, buprenorphine, codeine, and tramadol. Oral opioids have variable uptake in companion animals and should ideally be used in conjunction with other medications. Common side effects of opioids are nausea/vomiting, constipation, panting, and altered mental state/dysphoria.

NMDA antagonists: Low doses of NMDA antagonists are often used to decrease central sensitization or “wind up” pain in oncology patients. These can be especially helpful in highly painful animals with nerve or bone pain such as osteosar- coma patients. This class of drugs includes medications such as ketamine and amantadine and are safe in most patients. Side effects may include elevated heart rate, elevated blood pressure, increased salivation, and pupil dilation, but these effects are minimized at lower doses.

176 Alpha-2 adrenergic agonist: Dexmedetomidine and medetomidine have been used as part of sedation protocols for years, but they also have the added effect of analgesia both by acting on the alpha-2 receptors and by synergistic effects with opioid administration. Constant rate infusions can be added as part of a multimodal analgesia protocol in moderate to severe pain management. Side effects may include sedation, vasoconstriction, decreased cardiac output, bradycardia, AV block, nausea, hyperglycemia, and polyuria.

Anticonvulsants: Gabapentin has become a widely used medication in veterinary management especially for the treat- ment of chronic or neuropathic pain. Side effects of gabapentin are rare and include mild ataxia and sedation. Long-term therapy does not appear to worsen side effects. Pregabalin is a lesser used anticonvulsant that has a higher potency but has been studied less in veterinary medicine.

Tricyclic antidepressants: These medications block the reuptake of norepinephrine and serotonin, allowing these mol- ecules to remain present and act on pathways that modulate pain transmission. These medications include amitriptyline, clomipramine, and imipramine. Studies on safety and efficacy in veterinary medicine are currently limited.

Common comorbidities in oncology patients Oncology patients presenting for surgery often have one or more comorbidities or paraneoplastic syndromes that need to be taken into consideration by the anesthetist. Tumors can produce and release several biologically active substances that can be more deleterious than the original tumor. Examples of paraneoplastic syndromes include cancer anorexia- cachexia syndrome (CACS), fever, anemia, thrombocytopenia, hyperproteinemia, coagulation disorders, hypoglycemia, hypercalcemia, and other general manifestations.

Cancer Anorexia-Cachexia Syndromes: This is the most common paraneoplastic disorder in veterinary patients. CACS is characterized by nausea, anorexia, and weight loss that ultimately lead to severe body fat depletion and muscle wasting. Due to a body composition with less fat and muscle, higher circulating levels of drugs may be present. Drug dosages may need to be altered to avoid overdose, prolonged recoveries, or rough recoveries. These patients may be more prone to hypothermia during the anesthetic period, so the anesthetist should decrease heat loss by insulating from cold surfaces, wrapping the patient, using warm water blankets, and/or using other active heating.

Fever: This occurs in the oncology patient either due to the production and release of pyrogenic cytokines by the tumor or from the patient’s immune system. Severe hyperthermia can cause and increased metabolic rate and oxygen consump- tion that can lead to dysfunction of multiple organ systems. This may include acute renal failure, disseminated intravas- cular coagulation, or arrhythmias. This may also cause faster drug metabolism and need for increased inhalant anesthetic concentrations. Patients should be treated for hyperthermia prior to anesthetic induction if possible.

Anemia: In cases where the tumor is causing hemorrhage or hematopoietic suppression, removal of the tumor may be necessary to improve anemia. Acute or severe anemia should be stabilized prior to anesthesia with a whole or packed red blood cell transfusion, especially if hypovolemia or hypoperfusion are a concern. Preoxygenation is vital in these patients to maximize oxygenation and guarantee optimal perfusion to tissues throughout the procedure.

Thrombocytopenia: Surgery and anesthesia may be necessary to remove malignancies triggering thrombocytopenia. The use of cytotoxic drugs may increase risk of thrombocytopenia in oncology patients. A coagulation profile (PT, PTT) can assess clotting function prior to surgery. If indicated a plasma transfusion may be necessary to prevent excessive hemor- rhage intraoperatively.

Hyperproteinemia: Patients with multiple myeloma, lymphoma, or leukemia may have concurrent hyperproteinemia. Bleeding disorders may be present in these patients due to poor platelet aggregation. Other patients may present with hyperviscosity and blood sludging, which can lead to tissue hypoxia, ocular changes, CNS deficits, cardiac disease, or multiorgan failure. In severe cases plasmaphoresis may be necessary prior to anesthesia.

177 Hypercalcemia: Bone reabsorption by osteoclasts and concurrent release of circulating calcium may lead to hypercalcemia in oncology patients. Other diseases can cause hypercalcemia including hypoadrenocorticism and renal failure, so those diseases should be ruled out preoperatively. Signs of hypercalcemia include polyuria, polydipsia, muscle tremors, weak- ness, vomiting, bradycardia, stupor, and/or coma.

Cardiac arrhythmias: Changes in intracellular signaling pathways may lead to the presence of cardiac arrhythmias in the cancer patient. The most common cardiac arrhythmias seen are premature ventricular complexes and atrial fibrillation. All oncology patients should have an ECG performed prior to anesthesia, and proper treatment initiated if an arrhyth- mia is present and the patient is clinical.

Procedural complications Cancer can be present in almost any part of the body, so surgical oncology procedures can be widely variable. Some com- plex procedures that can be performed to remove primary tumors include splenectomies, liver lobectomies, amputation, partial pancreatectomies, mandibulectomies, thyroidectomies, and anal sacculectomies to name a few. Management of these procedures should be case-based, and protocols tailored to each patient. Some splenectomies or liver lobectomies may require blood product administration, while others may be straightforward and low risk. Some thyroidectomies may be quick procedures, while others have a risk of swelling, respiratory distress, or hemorrhage. It is the responsibility of the anesthetist to understand potential complications for each procedure and be prepared for them prior to surgery.

Oncology patients can present many challenges that the anesthetist should be prepared for prior to surgery. Pain manage- ment in these patients can be tricky, they can have multiple comorbidities, and the surgical procedures may be compli- cated. Thorough workups, knowledge of multimodal analgesia, understanding of paraneoplastic syndromes, and famil- iarity with necessary surgical procedures are key elements in successful management of anesthesia for oncology patients.

References 1. Fan TM. Cancer Patients. In: Grimm KA, Lamont LA, Tranquilli WJ, Greene SA, Robertson SA, editors. Veteri- nary Anesthesia and Analgesia: The Fifth Edition of Lumb and Jones. 5th ed. Ames: Wiley-Blackwell; 2015, pp. 993-998. 2. Salazar V. Neoplastic Disease. In: Johnson RA, Snyder LB, editors. Canine and Feline Anesthesia and Co-Existing Disease. 1st ed. Ames: Wiley-Blackwell; 2015, pp. 264-290. 3. John WJ, Patchell RA, Foon KA. Paraneoplastic syndromes. In: DeVita VT, Hellman S, Rosenberg SA, editors. Cancer: Principles and Practice of Oncology. 5th ed. Philadelphia: Lippincott-Raven; 1997. pp. 2397–2422.

178 TECHNICIAN OR THERAPIST: COMMUNICATION THROUGH GRIEF Danielle DeCormier, LVT, VTS (Oncology), CCFP

Introduction Veterinary medicine is filled with ups and downs. While many of us are trained on how to performs skills and discuss cases, it is only recently that communication skills have been looked at. With compassion fatigue and burn out plaguing the industry, it is important to know what they are, and how they change our interactions. It is also important to recognize grief and the way it impacts understanding. This session delves into defining grief, as well as anticipatory grief, and how to work through it. It also goes through various types of communication and how to get your message through.

Grief There has been a lot of debate around the various stages of grief; what they are and do they even exist. Five stages used to be the standard, however, there are now commonly seven recognized. Rather than adding two separate stages, it just breaks down the original ones further. The five stages discussed here are Shock and denial, pain and guilt, anger and bargaining, depression, and acceptance. These stages are not milestones and do not go in any specific order. Someone can start with any of them and hit all or even none of them. There is also no length of time, or anything preventing circling back through them.

Shock and denial are that state of disbelief. A person can seem numb. This is a way of their brain protecting them from absorbing more than they can handle. It is not a state of uncaring. Pain and guilt are the acute manifestation of physical plain. This is where internal blame takes hold. Anger and bargaining is the opposite. This is where outward blame is displayed. This is often very intense and can often be mistaken for just being a difficult client in the veterinary world. Bargaining generally means trying to make deal with God in times of grief. Depression is usually the longest stage and the most socially acceptable because it is expected. This encompasses sadness and loneliness. The final stage is acceptance. This is when people begin to see hope for the future and what their life will look like after the loss. It is a good idea to remember that the other stages can resurface well after a person has gone through acceptance.

In veterinary medicine, our clients can experience grief in any number of situations. In many of these, the client is not prepared for decisions that have to be made or able to understand what is happening. In situations that do not involve an immediate loss of life, anticipatory grief can begin. This type of grief happens when an owner is confronted with the mortality of their pet. In other words, their pet may die from this condition. The stages are the same for this as with the actual loss of their pet and can be just as intense. At this moment, a grieving client can easily look like a difficult client. All of the blame, anger, and denial that is seen can be attributed to grief. This is not to excuse truly difficult clients or their bad behavior, but to provide insight on how to proceed.

Communication Cycle There is a communication cycle that happens with every single thing that we communicate, including medical information about a pet. It starts with the sender, who decides to send the message and how to send it. This can be done in person, in writing, or over the phone. Outside factors come into play here with noise levels and nonverbal signals the sender is emitting. Then it gets to the receiver. They begin processing the message based on their knowledge and understanding and recall previous messages they have received regarding that information, from any life experience they have. Then, based on this new knowledge and understanding, they respond and restart the cycle as the sender.

Grief changes everything about this cycle. It interferes with every step. It affects brain chemistry, memories, and even focus. They are distracted and may not have heard things correctly or completely. They may not be watching for nonverbal cues or even able to see them, depending on the form the message was sent in. Their ability to process information is completely different because the grief becomes all-encompassing in their brain. There was a study done on people making decisions about treatment of family members in the ICU, and it showed that grief worsened the ability to make those decisions.

179 Types of communication There are 2 types of communication: verbal and nonverbal. However, nonverbal has a subsection (paraverbal) made up of just tone of voice. A majority of communication is nonverbal, including body language, which accounts for 55%. Paraverbal, or tone of voice, is 38%. This leaves verbal at just 7%.

Nonverbal communication is not just body language though. It is also facial expression, eye contact, speech rate and volume, and tone of voice. The barriers to being effective in communicating this way are personal stress, distractions, environmental factors, including delivering more messages over the phone or via email. The ways to combat this are the 3 A’s – awareness, attentiveness, and acknowledgement. Be aware of what signals you are sending and the ones you are receiving. Be attentive and focus on the case immediately in front of you. Acknowledge what is being sent back, especially if it is clear your message is being received incorrectly. Pay special attention to surroundings, a decrease in face-to-face interactions, and the new learning curve associated with everyone wearing masks.

Verbal communication is much less intensive. This is literally the words we use. The barriers here are vocabulary, the knowledge gap between the sender and receiver, and what perception a certain word choice can convey. The 3 A’s are also extremely useful here. Be aware if you are using complicated medical words versus common language. Pay attention to how your words are being received, and acknowledge their understanding or misunderstanding. Take note of the fact that there are more surroundings and less nonverbal communication happening. Word choice is much more important now without those other cues.

Compassion Fatigue In veterinary medicine, staff are exposed to intense medical decisions and grieving clients constantly. This can lead to compassion fatigue, with is absorbing trauma and the emotional stresses of others creating secondary traumatic stress. This is caused by the work that is done. This is from the stress of being a care-giver. Some signs or symptoms are a lack of empathy, exhaustion, anger or anxiety, excessive complaining, and physical ailments. This can be treated with appropriate self-care, a vacation, and sometimes requires professional help. Self-care is the biggest method of prevention. This is not to be confused with burnout.

Burnout Burnout is exhaustion. This is caused from our work environment: workload, work-life balance, lack of support, and stress. This is what a majority of veterinary professionals go through, especially now. Signs of this are anger and impatience, dehumanizing others, substance abuse, physical ailments, withdrawal. Treatment can be complicated and recovery is not guaranteed. It includes a support system, self-care, communication, even professional help or a career change. Prevention is also more involved than with compassion fatigue because a simple vacation does not often work. One needs to have recognition of it, communication, awareness and then must enact change and avoid toxicity.

References • Ellis, C. (2011). Pet parents: a journey through unconditional love and grief. Bloomington, IN: IUniverse. • Ayl, K. (2013). When helping hurts: compassion fatigue in the veterinary care professional. Lakewood, CO: AAHA Press. • Assess Your Wellness. (n.d.). Retrieved January 27, 2018, from https://www.avma.org/ProfessionalDevelopment/ Personal/PeerAndWellness/Pages/assess-your-wellness.aspx • MacEwen, E. G., Vail, D. M., & Page, R. L. (2013). Withrow & MacEwen’s Small Animal Clinical Oncology. St Louis: Elsevier-Saunders. • Moore, A. S., & Frimberger, A. E. (2010). Oncology for veterinary technicians and nurses. Ames, IA: Wiley-Blackwell.

180 Continuing Education Information

The number of CE hours that you will earn at this conference is dependent on the presentations you attend live during the actual conference as well as on demand afterwards.

New York-based Veterinarians and Technicians The 2020 VCS Annual Conference has received the Certificate of Approval as a Sponsor of Continuing Education for Veterinarians and Veterinary Technicians from the State Board for Veterinary Medicine.

Texas-Licensed Veterinarians If you are licensed to practice in Texas, we can provide you with the appropriate CE form indicate sessions in which you attended. Please request that form at [email protected].

All sessions run in partnership with the Missouri Veterinary Medical Association (MVMA) and have been approved as MVMA continuing education credit. Participants should be aware that some boards have limitations on the number of hours accepted in certain categories and/or restrictions on certain methods of delivery.

Abstract Information The abstracts published in these proceedings should be treated as peronal communication and cited only as such with the consent of the author. Copyrights of this material belong exclusively to the authors and may not be reproduced without their consent. Abstracts were selected by the VCS Executive Committee on the basis of adherence to written guidelines and scientific merit of the abstract material.

181 Author Index

Name Page # Name Page # Abadie, Jérôme...... 76, 77 Bygott, Thomas...... 63 Aguirra, Lucien Roberta...... 109 Byron, Sara...... 122 Al-Nadaf, Sami...... 58 Cadieu, Edouard...... 76, 77 Albertini, Mark...... 60 Callejo, Andrea...... 103 Allen, Irving...... 59 Campbell, Justine...... 116 Andre, Catherine...... 77 Cannon, Claire...... 136 Ansai, Shinichi...... 102 Carossino, Mariano...... 132 Asada, Hajime...... 74 Carroll, Jennifer...... 59 Asano, Kazushi...... 69 Carter, Gabrielle...... 57 Atsushi, Maeda...... 107 Cascio, Matthew...... 87 Aurisicchio, Luigi...... 75 Cebrián Pinar, Elisa...... 92 Avery, Anne...... 133 Chadsey, Laura...... 118 Aviram, Gal...... 83 Chambers, James...... 125 Ayers, Jordan...... 87 Chibuk, Jason...... 113 Baham, Shelby...... 111 Childress, Michael...... 131 Balasuriya, Udeni...... 132 Choi, Eunju...... 138 Banovich, Kathryn...... 122 Chorny, Ilya...... 113 Barry, Sabrina...... 59 Chuang, Han-Yu...... 122 Bechtel, Sandra...... 87, 130 Church, Molly...... 123 Bednarz, Bryan...... 60 Cianciolo, Rachel...... 119 Berger, Erika...... 86 Clifford, Craig...... 58, 86 Bergman, Philip...... 86 Colombo, Jucimara...... 121 Beyers, Matthew...... 128 Conforti, Antonella...... 75 Billhymer, Audrey...... 96 Cook, Matthew...... 58, 118, 119 Boateng, Martin...... 122 Cortés-Hinojosa, Galaxia...... 87, 130 Bohannan, Zack...... 72 Costa, Victoria...... 117, 132 Borges, Barbara...... 109 Coutermarsh-Ott, Sheryl...... 59 Borrego Massó, Juan Francisco...... 92, 112 Curran, Kaitlin...... 119, 129 Botherel, Nadine...... 77 Dagli, Maria Lucia...... 95 Boudreaux, Bonnie...... 117 Dank, Gillian...... 83 Boye, Pierre...... 68 Dark, Michael...... 91 Bowal, Jacqueline...... 134 David, Emmanuelle...... 68 Bradley, Abbey...... 88 De Fornel, Paulie...... 77 Brady, Rachel...... 138 De Mello Souza, Carlos...... 58 Brandt, Sabine...... 78 Deguchi, Tatsuya...... 89 Brehm, Amanda...... 61 Denis, Jerome Alexandre...... 77 Briones, Natalia...... 122 DePauw, Taylor...... 70 Brown, Graham...... 116 Dervisis, Nick...... 59 Brown, Megan...... 119 DeTaboada, Luis...... 88 Bryan, Jeffrey...... 90 Devauchelle, Patrick...... 76, 77 Burbano, Rommel Mario...... 109 Deveau, Michael...... 61 Burton, Jenna...... 138 Dewsbury, Nathan...... 63, 85

182 Author Index Author Index

Name Page # Name Page # Diaz, Sandra...... 118 Han, James Enyang...... 72 Diniz, Pedro...... 57 Haney, Siobhan...... 97 Dolan, Christopher...... 63 Harada, Kei...... 74 Donnelly, Lindsay...... 90 Harada, Maria Lucia...... 109 Dorso, Letitia...... 77 Hardat, Nathalie...... 85 Doty, Andria...... 130 Harris, Nicolette...... 100 Elias, Lourdes...... 57 Harvey, Garrett...... 126 Engelien, Julia...... 91 Haworth, David...... 122 Espinosa, José...... 103 Hecht, Toby...... 128 Facchetti Vinhaes Assumpcao, Anna Luiza...... 84 Hedan, Benoit...... 76, 77 Facista, Salvatore...... 122 Hendricks, William...... 122 Feenstra, Laurien...... 66 Hernandez, Oscar...... 87, 91 Feldhaeusser, Brittany...... 62 Hernandez, Reinier...... 60 Fernandez, Elmer...... 72 Herrera, Chamisa...... 58 Flesner, Brian...... 90 Herzog, Marielle...... 63, 85 Flory, Andi...... 113 Hirata, Akihiro...... 127 Fonseca-Alves, Carlos...... 108 Hiroki, Sakai...... 107 Francois, Romain...... 77 Hocker, Samuel...... 64, 93 Frasca, Salvatore...... 91 Holtvoigt, Lauren...... 113 Fredrickson, Kirsha...... 97 Horwitz, Harrison...... 72 Fries, Ryan...... 98 Hosaka, Soshi...... 114, 115 Fukushima, Ushio...... 102 Hosoya, Kenji...... 89 Fukuyama, Yasuhiro...... 102, 114, 115 Hume, Kelly...... 135 Fulkerson, Christopher...... 137 Husbands, Brian...... 58, 129 García de la Virgen, Miguel...... 92, 112 Hutchison, Shana...... 130 Gasparini, Molly...... 119 Impellizeri, Joseph...... 75 Gasson, Shelby...... 119 Inkol, Jordon...... 64, 93 Gedney, Allison...... 120 Inoue, Marina...... 82 Gehring, Ronette...... 66 Irie, Mitsuhiro...... 74 Gieger, Tracy...... 97 Ishigaki, Kumiko...... 69 Gillette, Robert...... 100 Iwasaki, Ryota...... 104, 110 Godoy, Bianca...... 121 Jackson, Kirsten...... 130 Gogal, Robert...... 62 Jannasch, Amber...... 137 Goto, Sho...... 104, 110 Jansenss, Brandan...... 119 Goto-Koshino, Yuko...... 125 Jarvis, Jill...... 61, 63, 85 Green, Eric...... 118 Jegatheeson, Selvi...... 136 Griffin, Lynn...... 119 Jennings, Ryan...... 118 Grosu, Daniel...... 113 Jensen, Mark...... 128 Grudzinski, Joseph...... 60 Jindra, Christoph...... 78 Gur, Assaf...... 83 JIa, Shuai...... 84 Hainisch, Edmund K...... 78 Johannes, Chad...... 86, 116 Hamilton, MJ...... 88 Jones, Pamela...... 116

183 Author Index

Name Page # Name Page # Jozwiak, Caitlin...... 123 Lundberg, Alycen...... 96 Kamishina, Hiroaki...... 82 Madarame, Hiroo...... 102 Kawarai, Shinpei...... 102 Maeda, Atsushi...... 104 Kayanuma, Hideki...... 114, 115 Magee, Kara...... 60 Keepman, Samuel...... 56, 119 Maitz, Charles...... 90 Kelly, Terry...... 63, 85 Marescaux, Laurent...... 68 Kelsey, Krista...... 97 Marsh, Ian...... 60 Khammanivong, Ali...... 70 Martins, Rene...... 120 Kim, Erika...... 128 Maruo, Takuya...... 102, 114, 115 Kim, Sangho...... 89 Matsumoto, Yuki...... 74 Kim, Se-hoon...... 106 Mauldin, Glenna...... 101 Kisseberth, William...... 118 Mauldin, Guy...... 101 Klahn, Shawna...... 59 Maxwell, Elizabeth...... 119 Knapp, Deborah...... 128 McCain, Robyn...... 131, 137 Knipp, Gregory...... 137 McEntee, Margaret...... 97 Kobayashi, Priscila...... 108 McMillan, Sarah...... 58 Kobayashi, Tetsuya...... 74 Melton, Tammy...... 88 Kohei, Yamada...... 107 Merkle, Jennifer...... 111 Koo, Jamin...... 72 Michel, Gaetan...... 63, 85 Kousoulas, Konstantin...... 111 Michel, Kathryn...... 123 Krick, Erika...... 58, 120 Miller, Lisa...... 88 Krupa, Ada...... 66 Miller, Tasha...... 61, 63, 85 Kubicek, Lyndsay...... 97 Milner, Rowan...... 87, 130 Kuehnl, Ashley...... 84 Modiano, Jaime...... 70 Lainetti, Patricia...... 108 Moeller, Cambri...... 111, 117 Lambert, Lindsay...... 126 Moore, Anthony...... 119 Lammens, Tim...... 66 Moore, Peter...... 118 Langohr, Ingeborg...... 132 Mori, Takashi...... 104, 110 Laufer-Amorim, Renee...... 108 Morris, Zachary...... 60 Laver, Travis...... 62 Moschetta-Pinheiro, Marina...... 121 Le Bot, Ronan...... 68 Mukherjee, Amit...... 128 LeBlanc, Amy...... 128 Murakami, Mami...... 104 Lee, Hye-Ryeon...... 72 Murase, Yusuke...... 89 Leeper, Haley...... 58 Murray, Caroline...... 58 Leis-Filho, Antonio...... 108 Musk, Philip...... 128 Lejeune, Amandine...... 87, 91, 130 Musser, Margaret...... 86 Lembcke, Luis...... 137 Muster, Thomas...... 78 Lewis, Ben...... 126 Mutsaers, Anthony...... 64, 93 Lim, Sungwon...... 72 Nagamine, Marcia...... 95 Liu, Chin-Chi...... 111 Nagata, Koichi...... 62 Looper, Jayme...... 111 Nagumo, Takahiro...... 69 Lopes, Christina...... 126 Nakamoto, Yuya...... 82

184 Author Index

Name Page # Name Page # Nemoto, Yuki...... 114, 115 Reinhart, Jennifer...... 98 Nishida, Shuhei...... 127 Reising, Abby...... 90 Nishiyama, Yuta...... 102, 114, 115 Reyes, Rothman...... 131 Nisii, Naohito...... 127 Richardson, Danielle...... 58 Noguchi, Shunsuke...... 82, 94, 105 Rinno, Jun...... 125 Nolan, Michael...... 97 Riva, Alberto...... 87 Novais, Adriana...... 121 Rodrigues, Lucas...... 126 Ogata, Niwako...... 137 Ronan, Le Bot...... 74 Ogilvie, Gregory...... 57 Ruslander, David...... 97 Ohmi, Aki...... 125 Ruth, Jeffery...... 59 Ohno, Koichi...... 125 Ryutaro, Yoshikawa...... 107 Okumura, Masahiro...... 89 Saba, Corey...... 97 Orito, Kensuke...... 114 Sahay, Bikash...... 87, 130 Otridge, John...... 128 Sakai, Hiroki...... 104, 127 Palombo, Fabio...... 75 Salah, Pascale...... 120 Pan, Xuan...... 84 Salute, Marc...... 87 Parchment, Ralph...... 128 Salvatori, Erika...... 75 Park, Stanley...... 72 Satoru, Kohgo...... 107 Parshley, Lisa...... 88 Scavelli, Diane...... 129 Pellin, Mackenzie...... 56, 58, 129 Scavello, Heather...... 120 Pereira, Washington Luis...... 109 Selmic, Laura...... 119 Pereira Rivera, Constanza...... 92 Selting, Kimberly...... 96, 98 Pérez, Valentín...... 103 Seo, Kyoung-won...... 106 Pinard, Christopher...... 64 Serres, Francois...... 68 Pinheiro, Danilo...... 109 Shiomitsu, Keijiro...... 87, 91 Poirier, Valerie...... 134 Shirota, Kinji...... 102 Polk, Arthur...... 113 Siewert, Jacob...... 129 Polley, Amanda...... 72 Singh, Anju...... 128 Polliack, Gabriel...... 83 Skorupski, Katherine...... 58, 138 Poon, Andrew...... 64 Soileau, Aimee...... 117, 132 Post, Gerald...... 126 Sommers, Connie...... 128 Prinsen, Berthil...... 66 Sondel, Paul...... 60 Proulx, David...... 57 Sousa, Raissa...... 109 Prouteau, Anais...... 77 Souza, Fabiana...... 108 Pudupakam, Raghavendra...... 72 Stegelmeier, Ashley...... 64 Pyuen, Alex...... 133 Stunk, Rebekah...... 100 Quick, Cally...... 111 Subramanian, Ramesh...... 111 Rankin, Wendy...... 72 Sunaga, Takafumi...... 89 Rault, Mélanie...... 76 Sung, Koangyong...... 89 Rebhun, Robert...... 138 Swartzfager, Deanna...... 72 Reddell, Paul...... 116 Takashi, Mori...... 107 Regan, Daniel...... 133 Tallant, Colt...... 122

185 Author Index

Name Page # Name Page # Tamura, Kei...... 69 Withers, Sita...... 111, 117, 132 Tanaka, Miho...... 125 Wittenburg, Luke...... 98 Tanaka, Toshiyuki...... 94 Wolscheck, Markus...... 78 Tanimoto, Nanami...... 94 Wong, Shukmei...... 122 Teddy, Lucy...... 135 Wood, Darren...... 64 Terrell, Jason...... 63, 85 Wood, Geoffrey...... 64 Teske, Erik...... 66 Woods, J Paul...... 64 Thamm, Douglas...... 72, 133 Woodward, Andrew...... 136 Tierny, Dominque...... 68 Wouda, Raelene...... 119 Toledo, Gabriela...... 95 Xi Qi, Nicholas Seah...... 72 Tomiyasu, Hirataka...... 74, 125 Yamazaki, Hiroki...... 94, 105 Tomohiro, Sawadaishi...... 107 Yoda, Shinichiro...... 114 Tran Hoang, Christine...... 96 Yoneji, Kensuke...... 127 Tripp, Chelsea...... 86, 88 Yoneji, Wakana...... 127 Tsang, Josephine...... 72 Yoshida, Orie...... 69 Tsuboi, Masaya...... 125 Yoshihito, Ueno...... 107 Tsujimoto, Hajime...... 125 Yoshikawa, Hiroto...... 97 Tu, Chantal...... 72 Yoshizaki, Kyoko...... 127 Tullius, Jeri...... 131 Zismann, Victoria...... 122 Tuohy, Joanne...... 59 Zuber, Max...... 136 Turatsinze, Jean-Valery...... 85 Zuccari, Debora...... 121 Turek, Michelle...... 60 Uchida, Kazuyuki...... 125 Ueno, Hirona...... 115 Vail, David...... 60, 134 Vallejo, Raquel...... 103 Van Geijlswijk, Inge...... 66 Vazquez-Sanchez, Eliza...... 67 Vázquez, Sergio...... 103 Veluvolu, Sridhar...... 119 Vernau, William...... 118 Vitt, Jordan...... 98 Wada, Yusuke...... 94, 105 Wallace, Gabrielle...... 98 Warner, Carolyn...... 100 Warrier, Manisha...... 122 Watanabe, Kei...... 74 Weichert, Jamey...... 60 Whitsett, Timothy...... 122 Willcox, Jennifer...... 58, 138 Wilson-Robles, Heather...... 61, 63, 85

186