Second

World Veterinary
Cancer Congress

Proceedings

1st - 3rd March 2012

Hôtel Pullman Paris Bercy

Paris, France
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CONGRESS COMMITTEE

ESVONC
Malcolm J Brearley, President
Johan de Vos, Treasurer
Jacqueline de Zeeuw CEO

VCS
Ruthanne Chun, President
Sandi Strother, CEO
Laura Garrett, President-Elect
Barbara McGehee

Local Committee
Patrick Devauchelle
Pauline de Fornel-Thibaud
Claude Muller
Cecile Soyer

The organising committee wish to thank all those persons who helped review the submitted
abstracts, judged the Residents’ competitions, moved chairs, carried things and generally made the
smooth running of the conference possible.

Platinum sponsor
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Second World Veterinary Cancer Congress

1st - 3rd March 2012

Hôtel Pullman Paris Bercy
Paris, France

Day 1 Thursday 1st March

08:40 WELCOME
GENERAL ABSTRACTS Page
09:12 Jolle Kirpensteijn Radioactive microbrachytherapy for head and neck tumors in dogs
32
and cats
09:24 Valerie J Poirier Accelerated hypofractionated radiation therapy for the treatment
33
of feline oral squamous cell carcinoma: a pilot study
09:36 Catie Mcdonald Response rate and duration associated with a 4Gy 5 fraction
34
palliative radiation protocol
09:48 Sandra M Axiak Phase 1 clinical trial of intratumorally administered radioactive
35
gold nano-particles in canine prostatic carcinoma
10:00 Michelle Turek Current clinical practice in target volume and dose specification
36
reporting in veterinary radiation oncology
10:12 Jessica Lawrence Investigation of masitinib on radiosensitivity of feline injection site
37
sarcoma cells
10:25-10:55 Coffee and Posters
11:00 Lisa Pang Canine glioma cancer stem cells have global defects in DNA
38
damage pathways induced by ionising radiation
11:12 Stuart C. Helfand SRC blockade mediates potent inhibitory effects on growth and
39
survival of canine hemangiosarcoma
11:24 Richard Elders Development of recombinant canine IgE fc-based immunotherapy
40
for canine mast cell tumours.
11:36 Joanna Morris Transgenic models reveal delayed development and oncogenic
41
effects in mouse mammary glands over expressing RUNX2
11:48 Alejandro Epithelial to mesenchymal transition in canine breast cancer
42
Cervantes Arias
12:00 Catherine Ibisch Outcome of triple negative mammary carcinomas in dogs. 43
12:15-13:50 Lunch on your own and Posters
MINI-SYMPOSIUM FAMILIAL AND BREED-RELATED CANCERS
14:00 Prof Sir Bruce Common genetic variation, applications to cancer risk and
Ponder prevention in man 2
KEYNOTE LECTURE
14:50 Dr Catherine Andre Familial cancer in dogs
4
KEYNOTE LECTURE
15:40-16:10 Tea and Posters
THEMED ABSTRACTS
16:12 Jesus Aguirre- Whole genome mapping of anal sac gland carcinoma susceptibility
63
Hernandez loci in the English cocker spaniel
16:24 Jaime Modiano Heritable traits influence cell-intrinsic behaviour of sporadic canine
64
cancers
16:36 Malin Melin Identification of predisposing genetic risk factors in canine cancer 65
16:48 Johan De Vos Familial non-medullary thyroid carcinoma in the German pointer
66
longhair.
17:30 WELCOME Restaurant de l’Ecole Nationale Vétérinaire d’Alfort
Buses RECEPTION
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Day 2 Friday 2nd March

08:50 Introduction RESIDENTS’ ABSTRACTS
09:00 Andreia Santos The prognostic value of VEGFR2 in female dogs with mammary
10
gland tumours: a univariate survival study.
09:12 Floryne Buishand The GH/IGF-1 axis: a potential target for novel canine insulinoma
11
therapies
09:24 Cláudia Sofia Sequence variant spectrum of the feline TP53 gene in mammary
12
Baptista gland hyperplasia and neoplasia
09:36 David Sayag Valosin containing protein (VCP) is a potential novel serum marker
13
for cancer in dogs
09:48 Spela Bavcar Lomustine induced renal toxicity in dogs: a retrospective study
14
with a prospective follow-up.
10:00 Riccardo Finotello Preliminary study on a combined doxorubicin-dacarbazine
chemotherapeutic protocol for the adjuvant treatment of canine 15
hemangiosarcoma
10:12 Nele Van Den Steen Rectal lymphoma - a case series of twelve dogs 16
10:24 Lorella Maniscalco PDGF receptors: new targeted therapy in canine osteosarcoma 17
10:40-11:10 Coffee and Posters
11:12 James Warland The utility of staging in canine mast cell tumours 18
11:24 Nan Anti-angiogenesis and increased mural cell coverage by low-dose
Choisunirachon cyclophosphamide with piroxicam in canine malignant melanoma 19
xenografts.
11:36 Veronica Toxicity and pharmacokinetic profile of a new anticancer agent in
20
Kristiansen canine mammary cancer
11:48 Ksenia Lisitskaya The use of adjuvant protocols with docetaxel and doxorubicin in
21
cats with grade 3 mammary cancer.
12:00 Ana Laura Saraiva Expression of oestrogen and progesterone receptors on feline
22
endometrial adenocarcinomas
12:12 Tor Espen Classification of canine seminomas as classical or spermatocytic
23
Thorvaldsen using morphology and immunohistochemistry
12:25-13:50 Lunch on your own and Posters
14:00 Prof Ian Hart Biology of tumour invasion and metastasis; therapeutic targeting
5
KEYNOTE LECTURE of underlying mechanisms
RESIDENTS’ ABSTRACTS
15:00 Aric Frantz Cancer stem cells from three ontogenetically distinct canine
24
tumors have shared patterns of gene expression
15:12 Lisa Pritchard Role of Sulf1 and Sulf2 splice variants in canine mammary tumour
25
growth
15:24 Joanna Mucha Correlation between number of Gr1-positive myeloid precursor
cells, P-Stat3 expression and gene expression pattern in canine 26
mammary cancer metastasis
15:36 Krystal Claybrook- Patterns of Ctip2 immunoreactivity in feline oral squamous cell
27
Harris carcinoma
15:50-16:20 Tea and Posters
GENERAL ABSTRACTS
16:24 Marja K. De Jong Expression of Sfrp1 and activation of the Wnt-pathway in healthy
29
and affected adrenal glands of ferrets
16:36 Tsuyoshi Kadosawa Total cystectomy and uretero- urethral/preputial/vaginal
30
anastomosis in 23 dogs with transitional cell carcinoma of bladder
16:48 Enrico Pierluigi Electroporation enhances bleomycin efficacy in cats with advanced
31
Spugnini head and neck carcinoma
17:00 - ESVONC AGM Free evening!
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Day 3 Saturday 3rd March

GENERAL ABSTRACTS
08:48 Saskia Willenbrock Establishment of a new canine prostate carcinoma cell line with
44
complex karyotype changes involving polysomy-13
09:00 Sabine Brandt Evidence for a causal association of ECPV-2 with equine squamous
45
cell carcinoma
09:12 Ai Watabe The effect of surgical stress on regulatory T cells in dogs with
46
cancer
09:24 Ayako Kamida The effect of anti-Rankl antibody on canine osteosarcoma in a
47
xenograft mouse model
09:36 Lisa Pang Global gene expression analysis of canine osteosarcoma cancer
48
stem cells
10:00- Dr Luc Stockx, MD Interventional radiology in oncology
6
10:45 KEYNOTE LECTURE
10:50-11:20 Coffee
GENERAL ABSTRACTS
11:24 Joaquim Henriques Evaluation of a panel of different antibodies to assess B-cell
lineage by immunohistochemistry using a tissue microarray in 49
canine lymphoma
11:36 Mery Giantin Evaluation of c-Kit (CD117) Mutations, M-RNA and protein
expression in canine leukemia and lymphoma: might c-Kit 50
represent a therapeutical target?
11:48 Arianna Aricò Matrix metalloproteinases and vascular endothelial growth factor
51
in canine lymphohematopoietic malignancies
12:00 Joaquim Henriques Application of Han’s algorithm to classify diffuse large B-cell
52
lymphoma (DLBCI) in dogs
12:12 Irina Gramer The drug efflux carrier MDR1 influences efficiency and tolerability
53
of the cytostatic treatment of malignant lymphoma in dogs.
12:25-13:45 Lunch in the hotel
13:50- Dr Patricia Carrigan A picture is worth 10,000 Words; Next Generation of Companion
8
14:45 KEYNOTE LECTURE Diagnostics
GENERAL ABSTRACTS
14:48 Lise Nikolic Nielsen Cytokine profiling of Bernese mountain dogs with disseminated
54
histiocytic sarcoma
15:00 Gerard Rutteman Clinical trial in canine osteosarcoma with the oral biphosphonate
55
clodronate
15:12 Tine Janssens Treatment of dogs with carboplatin: excretion, contamination of
56
the environment and uptake by owners
15:24 Maria Carolina Efficiency and cytotoxicity of nano-particle mediated transfection
Duran Graeff methods for DNA expression plasmids as basis for cell therapeutic 57
approaches
15:40-16:10 Tea
16:12 Carolyn J. Henry The novel chemoprotectant drug, Tavocepttm, facilitates a
shortened cisplatin infusion protocol for treatment of cancer- 58
bearing dogs
16:24 François Serres Assessment of cardiac arrhythmia in dogs with cancer receiving
59
doxorubicin: a prospective Holter study of 20 dogs.
16:36 Federica Cavallo DNA vaccination against human CSPG4induces cross-reacting
antibody response in dogs with oral malignant melanoma 60
expressing CSPG4
16:48 Thora Johanna A new concept within localised cancer treatment
61
Jonasdottir
17:00 Close of Conference
19:00- Gala Dinner Bateaux Mouches (Boats on the Seine!!)
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A map!!

Hôtel Pullman Paris Bercy

1 rue de Libourne, 75012 PARIS, FRANCE

Tel : (+33)1-44673400

Another map
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Our Sponsors

Without our sponsors it would be impossible to organise a conference like this!

On behalf of WVCC, the Congress Committee wishes to express sincere gratitude.

To all Delegates – Please visit our sponsors and thank them personally!!

Platinum Sponsor
Merial
(see back cover)

Diamond Sponsor
Pfizer Animal Health

Gold Sponsor
AB Science

Silver Sponsor
BD Medical

European Society of Veterinary Oncology and Veterinary Cancer Society

also thanks to our exhibitors

Radiology Oncology Systems Inc., USA

Eurobio-AbCys, France

John Wiley & Sons Ltd, United Kingdom

Dechra Veterinary Products SAS, France

IDEXX Laboratories, France

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Our Diamond Sponsor
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Our Gold Sponsor:

Our Silver Sponsor:

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Disclaimer
Although every effort to ensure that the information available in these proceedings is
factually correct, the Congress Committee does not accept liability for any errors or
omissions. The Congress Committee does not endorse or accept any liability for views and
opinions expressed in any of the text or advertisements or any of the associated websites.

Scientific Abstracts
The abstracts as published in these proceedings have not been subjected to extensive
peer-review and therefore should not be quoted in publications. The abstracts may be
considered as Personal Communications and referenced as such, with permission of the
individual authors. Copyrights of the material belong exclusively to the authors and may
not be reproduced without their permission.

Authorship and affiliation

In the following abstracts the authoship has been reduced to the first 5 or 6; beyond this
‘et al’. The address given is that for the Presenting Author. Apologies to co-authors but
with listings of 10 - 15 authors each with a different address, the scientific data was in
danger of being lost from sight!
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Keynote Speakers
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Professor Sir Bruce Ponder

Li Ka Shing Professor of Oncology and Director of the Cancer
Research UK Cambridge Research Institute, UK

Sir Bruce Ponder trained in Internal Medicine at Cambridge, St.

Thomas’ Hospital and in Oncology at ICRF and Harvard, his research
has been in developmental biology and subsequently in cancer
genetics. He initiated one of the first familial cancer clinics in the UK
and his group identified ret as the predisposing gene for MEN 2 syndrome and established clinical
guidelines for the management of this syndrome. They were also closely involved in research that
led to the identification of the BRCA1 and BRCA2 genes for breast and ovarian cancer and in the
clinical management of cancer families. Most recently they led the first genome-wide association
study to identify common predisposing genes for breast cancer.

Professor Ponder has also led the development of a new cancer research centre in Cambridge over
the past ten years, culminating in his appointment as the first Director of the new Cancer Research
UK Cambridge Research Institute, which opened in 2007. He was awarded a knighthood in the 2008
New Year Honours List for services to medicine.

Common genetic variation, applications to cancer risk and prevention in man

Each of the common cancers shows a tendency to cluster in families. Most of this clustering has an
inherited basis. In some cancers, such as breast cancer, rare but strongly predisposing mutations in
genes such as BRCA1 and BRCA2 account for a part of the total inherited effect; but for every
cancer type at present the majority of the effect remains unexplained.

Almost certainly, the unexplained component of genetic predisposition is accounted for by the
combined effects of hundreds of different variants, which may be common or rare but are
individually small in their contribution. This is the ‘polygenic model’ of cancer susceptibility.
Common genetic variants of this sort can be searched for using genome-wide association studies.
Recent studies in breast and prostate cancer have identified common variants which account for an
additional roughly 10% and 25-30% respectively of the inherited genetic component of these
cancers.

There are three broad questions to be addressed.

(1) What types of genetic variant account for the still unexplained 70% or so of genetic
predisposition to these cancers, and how should we best search for them?
Here the current money is on rare variants, that are not easily detected by association studies
and that might be expected to be identified in the future by exome or whole genome sequencing.
However it is likely that there will be a long ‘tail’ to the distribution of polygenic effect sizes, with
many common and rare variants having effects too small to detect individually, So, to measure the
effects of these variants, we need to find ways to develop readouts of their combined effects,
possibly for example through gene expression signatures.

(2) Of what practical use are the variants that have been identified?
Here the most secure answer is that new genes will provide new insights into tumour biology
and hence possibly new targets for intervention. The less secure answer – but the one that has
attracted most attention and controversy - is that combinations of these variants will provide ‘risk
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profiles’ that will enable the stratification of the population into different classes of risk, and that
this will be useful to individuals, to public health authorities who wish to target screening
programmes to best effect, and to researchers assembling prospective cohorts for intervention
studies.

(3) There is also the question of applications to prevention.

Although, in principle, individual common genetic variants may make substantial contributions
to the overall cancer burden in the population (the ‘attributable fraction’ of breast cancer for the
common variant in the FGFR2 gene, for example, is about 16%), the practical difficulties of
designing interventions based on mechanism are significant. There is nevertheless a possibility that
the gene expression signatures of predisposition referred to above might be reduced to specific
networks or pathways that could provide targets, and biomarkers, for prevention.

I will examine these issues in the light of experience from breast and prostate cancer.

References

Pharoah PDP et al (2008). NEJM 358, 2796-2803.

Gustafson et al (2010). Science Translational Medicine 2, 26ra25.
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Dr Catherine André
Head of the Canine Genetics Group
Centre National de la Recherche Scientifique
University of Rennes, France

Catherine André, PhD is the Head of the Canine Genetics Group in the
CNRS Unit at the School of Medicine, Rennes, France, where she has
also served as a Staff Scientist. Dr. André earned her PhD in Molecular
Oncology at the University of Paris. She has contributed extensively to
the field of canine genomics as well as elucidated the molecular basis
of many canine diseases, including histiocytic sarcoma in Bernese
Mountain Dogs, X-linked PRA in Border Collies and epilepsy. Catherine is Lead Scientist for the
Melanoma Project of the LUPA Initiative funded by the EC to study the genetic basis of dog diseases
of relevance to human health.
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Professor Ian Hart, FMedSci

Emeritus Professor, Barts Cancer Institute, , University of London, UK

Prof Ian Hart gained his BVSc in 1972 and his Ph.D in the Department
of Veterinary Medicine at Bristol in 1976. He had a post-doctoral and
Staff Scientist positions in the Frederick Cancer Centre, USA before
returning to the UK as a Principal Scientist at the Imperial Cancer
Research Fund in 1983. He was awarded the Professorship of the
Richard Dimbleby Department of Cancer Research, St. Thomas's
Hospital, London, in 1993. Professor Hart, who is an FRCPath, was
presented in May 2000 with the highest honour that a non-medically qualified person can receive
from the Royal College of Physicians, an Honorary FRCP. Most recently until his retirement he was
Centre Lead for Tumour Biology at the CR-UK Barts Cancer Institute.

Biology of tumour invasion and metastasis; therapeutic targeting of underlying mechanisms

Tumour cell invasion and metastatic spread are the defining features of malignant cancers in all multicellular
animals. Following neoplastic transformation and initial cancer growth disseminating tumour cells must
break away from the primary tumour mass, invade locally with penetration of lymphatic vessels or small
capillaries, transit as single cells or multicellular emboli through the circulation, arrest, either actively or
passively, at secondary sites, extravasate at distant sites and grow to establish secondary colonies.
Elucidation of the underlying molecular basis of the interactions involved in these characteristics may well
provide potential targets for therapeutic interventions which will ameliorate or prevent these devastating
traits of neoplastic disease.

Interactions between the extracellular matrix and invading tumour cells are important determinants of
malignant behaviour and these interactions largely are mediated by the integrin adhesion receptors,
heterodimeric glycoproteins expressed at the cell surface which are capable of both ‘inside out’ and ‘outside
in’ signalling. How de novo expression of one such heterodimer, the epithelial cell specific avb6, acts as a
prognostic marker and identifier of a specific molecular subtype of particular cancers will be detailed.
Moreover how expression of avb6 contributes to tumour aggression (partially through TGF-β activation and
matrix metalloproteinase upregulation), and how it can be used both as an imaging molecule ( to detect
avb6-positive deposits by PET or SPECT analysis ) and as a potential therapeutic target will be discussed in
the context of breast and pancreatic cancer. These findings illustrate that increased understanding of the
molecular basis of metastasis pathogenesis serves as a basis for the development of novel targeted
therapies.
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Dr Luc Stockx
Department of Medical Imaging,
Hospital “Oost Limburg”, Genk, Belguim

Dr. Luc STOCKX obtained his medical degree in 1988. After finishing his
radiology training in 1991, he became head of the division of Interventional
Radiology in the University Hospital of Leuven (Belgium). In 2000, he left for
a position of Interventional Neuroradiologist in Ziekenhuis Oost Limburg in
Genk a large community hospital in the East of Belgium. Although his main
interest today is the endovascular treatment of vascular diseases in the
brain, he is also active in interventional oncology. Beside supportive and
palliative treatments like venous or bile duct stenting and catheter
management, he performs chemo- and radio embolisations of liver
tumours. He is Fellow of the Cardiovascular and Interventional Radiological
Society of Europe (CIRSE), and member of many other International
Interventional Radiological organisations. He is also Education Program
Director of Vascular Solutions at the Crossroads Institute (ABBOTT Vascular)
in Brussels. He is first author of numerous publications in peer reviewed journals and of several
chapters in Interventional Radiological books.

Interventional Radiology in Oncology

Interventional Radiology has long been offering cancer patients palliative treatment for the
complications of their disease or of the consequence of their treatment. In the last decade, it has
leapt forward to take a more active role in oncology, giving birth to a new subspeciality:
Interventional Oncology.

Perhaps the most common means by which IR can facilitate patient treatment is by the provision of
image guided central venous access. Central venous catheters (CVC) provide a means of
administering medications or parental nutrition. Complications related to surrounding structures or
malposition of the catheter occur less commonly when performed with image guidance than when
performed blindly or using external landmarks. In addition IR has the ability to diagnose and treat
complications related to CVC insertion like catheter-directed thrombolysis and stenting of catheter-
induced thrombotic obstructions or the percutaneous removal of fractured catheter segments.

IR can also play an active role in treating the tumours themselves, either to shrink or kill them.
There are many different ways that IR can achieve this.
The first group of treatment modalities consist of transarterial embolisation techniques. Selective
injection of an embolic agent alone (bland embolisation) is performed to interrupt the afferent
blood supply to the tumour thereby inducing hypoxia and inhibiting tumor growth. It can also be
used in conjunction with ablative treatments or prior to surgery in an effort to reduce operative
blood losses. Transarterial chemoembolisation (TACE) is a modification of the above technique,
usually applied to hepatic tumours. It consist of the selective injection of a chemotherapeutic agent
(mostly doxorubicin), bounded on (eg. drug eluting beads) or in combination with an embolic agent
in the hepatic artery. The advantage of this technique over systemic chemotherapy is that delivery
of the chemotherapeutic agent is targeted at the lesion allowing a higher local concentration of the
agent and lower systemic doses. Embolisation of the artery increases the dwell time of the
chemotherapeutic agent. Radioembolisation, a novel form of liver-directed brachytherapy is
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performed by the transarterial introduction of beta-radiation emitting radioisotopes into the

tumour mass by means of microspheres. Most experience is obtained with Yttrium 90. Beta
radiation has a very low penetration (approximately 2.5 mm in human tissues), thus its necrosing
effects are localized.

Local tumour ablation is an alterative method of achieving tumour control in early stage malignant
disease that are not candidates for resection. Radiofrequency ablation (RFA) includes
administration of electromagnetic energy in the radiofrequency range to a tumour by means of a
locally placed electrode connected to a energy source. Tissues immediately surrounding the
electrode tip are heated to temperatures in excess of 60 degrees Celsius with consequent thermal
damage and cell death. This treatment modality has gained acceptance as a method of managing
hepatic and lung malignant disease, with efficacy also described in the treatment of adrenal, renal
and skeletal lesions. Cryoablation results in cell death due to the application of subfreezing
temperatures, achieved by use of Argon gas under high pressure. Alternating cycles of freezing and
thawing results in cell death due to the associated mechanical stresses upon cell membranes. The
application of electromagnetic energy in the microwave range agitates water molecules in targeted
tissues, resulting in frictional heat and cell death via coagulation necrosis.

Beside its role in the treatment of the disease itself, IR still has an important role in the
management of the complications of cancer. Malignancy can induce dysfunction of many organs
and systems. A significant portion of these complications are reversible by minimally invasive IR
methods, thereby relieving symptoms, thus having a significant positive impact on quality of live. If
endoscopic treatment of malignant obstructive jaundice due to extrinsic compression or tumour
invasion fails, this problem often can be solved by percutaneous transhepatic biliary drainage (PBD)
or stent placement. Malignant ureteral compression due to extrinsic tumour compression,
retroperitoneal adenopathy or direct tumour invasion frequently requires urinary decompression
by means of a percutaneous nephrostomy (PCN). Obstruction of the superior vena cava by
compression of tumour or adenopathies and responsible for vena cava superior syndrome can be
treated with percutaneous stent placement. In case of venous thrombosis, catheter directed
thrombolysis can be performed
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Dr Patricia Carrigan
Director of the Translational Diagnostics Assay
Development, Ventana Medical Systems, USA

With a career dedicated to the discovery of early detection

cancer biomarkers and the development of diagnostic
assays, Dr Patricia Carrigan currently is the Director of the
Translational Diagnostics Assay Development laboratory for
Ventana Medical Systems, Inc, a member of the Roche
Group. Building consensus with parties on all levels and
translating complicated and technical concepts to diverse audiences, Dr. Carrigan now leads two
independent teams of scientists in developing robust prototype assays that are used as companion
diagnostics for drug enrolment or stratification for clinical trials in the United States and in Europe.

A picture is worth 10,000 Words; Next Generation of Companion Diagnostics

During the initial diagnosis of a patient’s tumor be it from humans, canines or felines, the amount
of biopsy tissue collected predetermines the amount of downstream immunohistochemistries , in
situ hybridizations, and other molecular assays that can be performed. As more and more
companion diagnostics are approved and show successful response rates and efficacy similar to
Zelboraf for BRAF V600E for late stage melanoma and Crizotinib for ALK in Non-Small Cell Lung
Cancer, the initial tissue diagnostic assay must be highly accurate, sensitive, robust, and
reproducible. In this presentation you will hear how Roche Tissue Diagnostics develops Companion
Diagnostic immunohistochemical assays that can be used in exploratory and early and late phase
clinical trials. These diagnostic assays use the most state of the art antibody detection chemistries
and high throughput automated platforms available on the market. In addition you will learn about
the next generation of panel companion diagnostic assays that allow for multiple biomarkers to be
examined on one tissue section simultaneously. With the use of Quantum Dots and unique hapten
conjugation strategies, complex biological questions are now being investigated with answers being
generated with the aid of digital pathology and complimentary companion algorithm development.
As more correlated analyses are performed and new connections and associations are made
between the numerous biomarkers found within the heterogeneous context of a tumor, new
treatment strategies or regimens will lend themselves as better options as treating on the basis of
one biomarker expression pattern may no longer be the best approach.
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RESIDENT ABSTRACTS

The Wim Misdorp Award aims to encourage members in the earlier years of their career in
veterinary oncology to pursue a period of scientific investigation in the field of veterinary oncology.

The award is to recognize outstanding contributions to the knowledge related to pathogenesis,

diagnosis, therapy, prevention, or control of animal tumour-diseases. It is given once yearly at the
ESVONC Annual General meeting to a veterinarian based upon his/her written abstract and oral
presentation of scientific data at the meeting. In addition to a Certificate this award covers the
travel and registration costs for the author to attend the Veterinary Cancer Society Annual Congress
in the USA and to present their work.

The Wim Misdorp Award is sponsored by Pfizer Animal Health.

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The prognostic value of VEGFR2 in female dogs with mammary gland tumours: a univariate
survival study.

Andreia Santos
(Andreia Santos, Célia Lopes, Carlos Frias, Fátima Gãrtner and Augusto deMatos)

Multidisciplinary Unit for Biomedical Research (UMIB) and Department of Veterinary Clinics of the
Biomedical Sciences Institute of Abel Salazar (ICBAS), University of Porto, Portugal

[email protected]

Introduction
Canine mammary tumours (CMTs) can be very challenging to prognosticate due to their
heterogeneity and variable biological behaviour. Tumour angiogenesis has been considered one of
the hallmarks of cancer progression; several human cancer studies demonstrated than angiogenic
factors, such as vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2), are
associated with patient outcome. However, there are no survival studies regarding the prognostic
value of VEGFR-2 in CMTs. Hence, this work aimed to evaluate, through univariate survival analysis,
the potential value of VEGFR-2 for CMTs prognosis.

Materials
An immunohistochemical analysis of VEGF and VEGFR-2 was performed in 85 malignant CMTs to
investigate their association with the development of metastasis and patient survival. Dogs were
submitted to a 2-year follow-up. The Kaplan-Meier method was used to construct the survival
curves.

Results
VEGF and VEGFR-2 were significantly associated to each other. VEGFR-2 was related with
histological type and TIMP-2 expression, and tended to be higher in tumours with high MIB-1
labelling index. Other clinical and histopathological factors such as tumour size, histological grade
and mode of growth were not related to VEGFR-2 expression. Both VEGF and VEGFR-2 were not
significantly associated with the development of nodal and distant metastasis. Although patients
with tumours with high VEGFR-2 tended to have reduced disease-free intervals, these angiogenic
factors were not statistical significant predictors of patient?s survival.

Conclusions
The results of this study suggest that, despite VEGFR-2 association with some malignant
characteristics of CMTs, it was not a strong prognosticator of patient outcome.
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The GH/IGF-I axis: a potential target for novel canine insulinoma therapies

Floryne Buishand
(Floryne Buishand, Jan Mol, Marja Kik and Jolle Kirpensteijn)

Faculty of Veterinary Medicine, Utrecht University, Netherlands

[email protected]

Introduction
Long-term prognosis after surgical resection of canine insulinoma (INS) is poor. Signs of
hypoglycaemia often recur soon after surgery because tumour tissue has only been resected
partially and/or functional (micro-)metastases were present. Identification of genes involved in
proliferation and metastasis of canine INS may allow development of new medical treatment
options.

Materials
Using quantitative RT-PCR, the expression of 16 target genes was compared between 10 primary
canine INS and their corresponding metastases. For 8/10 primary INS and metastases,
immunohistochemistry was performed on proteins of the GH/IGF-1 axis. Canine cDNA microarrays
were used to study the difference in gene expression between 1. primary INS (n=10) and their
corresponding metastases; 2. microdissected normal islets of Langerhans (n=6) and primary INS
(n=6).

Results
There was significantly higher expression of genes encoding for growth hormone (GH) and insulin-
like growth factor-1 (IGF-1) in metastases compared to their primary tumours.
Immunohistochemical examination revealed expression of GH, IGF-1 and GH receptor (GHR) in both
primary INS and metastases. Immunohistochemical staining for IGF-1 was significantly higher in
metastases compared to primary tumours. IGF-binding protein 1 was one of the most down-
regulated genes in metastases versus primary INS, and IGF-binding protein 2 was amongst the most
down-regulated genes in primary INS versus microdissected pancreatic islets.

Conclusions
It is hypothesised that therapeutic intervention with agents that specifically antagonise the GH/IGF-
1 axis, or members of their signalling cascade, might inhibit canine INS growth and prevent
outgrowth of remaining tumour tissue after surgery.
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Sequence variant spectrum of the feline TP53 gene in mammary gland hyperplasia and neoplasia

Cláudia Sofia Baptista

(Cláudia Sofia Baptista, Carlos Frias, Joana Santos, Jorge Ribeiro, Liliana Martins, et al)

Institute of Biomedical Sciences Abel Salazar - University of Porto (ICBAS-UP), Portugal

[email protected]

Introduction
In breast cancer (BC), TP53 mutations are associated with worse overall and disease-free survival,
independent of other risk factors, and have been implicated in resistance to anticancer therapies.
Our purpose was to isolate and screen the feline TP53 gene for sequence variations (SVs) detection
in 10 normal, 9 hyperplastic and 39 neoplastic cat mammary gland tissues.

Materials
Cat genomic DNA was extracted from samples and three ERBB2 DNA fragments were amplified,
sequenced and analyzed with three sets of primers covering exons 2-4, 5-6 and 7-9 (572bp, 359bp
and 657bp, respectively).

Results
In total, we observed 21 SVs. The four SVs identified in the coding region were synonymous. The
most polymorphic exon and intron were, respectively, 5 and 7. Three intronic SVs were only
observed in carcinomas, one in a fibroadenomatous change and two in control samples.

Conclusions
We present de novo TP53 SVs in cat mammary masses. We did not observe the SVs previously
reported in feline mammary tumours. None of the already mentioned hot spot SNPs/mutations
described in BC were identified in cat mammary samples, although a silent mutation was
recognized in a hot spot codon (cat codon 155 and human codon 163), suggesting that this DNA
region is a genomic fragile site. In this series of samples, TP53 revealed to be highly polymorphic,
mainly in the noncoding region, both in controls and mammary masses. However, in cat, further
studies are required to demonstrate, or not, that these SV may increase cancer susceptibility and
modify cancer phenotypes in TP53 mutation carriers.
 n d
2 WVCC 2012 - 13 –

Valosin Containing Protein (VCP) is a potential novel serum marker for cancer in dogs

David Sayag
(David Sayag, Alexandre Boyer, Christian Bédard, Derek Boerboom and Marie-Éve Nadeau, )

Faculty of Veterinary Medicine - University of Montreal, Saint-Hyacinthe, Canada

[email protected]

Introduction
Although serum biomarkers are often used in human oncology as diagnostic tools or to monitor for
disease recurrence, there are few validated serum cancer markers available in veterinary medicine.
Recently, our laboratory has identified a new serum biomarker, Valosin Containing Protein (VCP), in
humans with malignancies. Our research suggests that VCP is a highly sensitive indicator of
malignancy, and is often increased in cancer patients who do not otherwise display increased levels
of the conventional serum tumor markers for their cancer type (CA125, CEA, or CA15.3). We
therefore hypothesized that VCP could be a valuable serum marker for canine cancer.

Materials
Serum from healthy (n = 7) and cancer-bearing (n = 16) dogs was assessed for VCP expression by
immunoblot analysis. Signal was quantified by densitometry.

Results
The assay detected serum levels of VCP over 12-fold higher in cancer-bearing dogs compared to
healthy dogs (P < 0.02). Furthermore, serum levels of VCP were on average 11-fold, 17-fold and 18-
fold higher in dogs with, respectively, histiocytic sarcoma (n = 2), lymphoma (n = 3) and various
carcinomas (n = 6) relative to normal dogs.

Conclusions
VCP seems to be a marker of interest for cancer in dogs, and could represent a valuable new tool
for diagnosis and follow-up in the clinical setting. Analyses of larger cohorts are taking place in
order to determine the sensitivity, specificity and predictive values of VCP measurement for various
forms and stages of cancer.
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- 14 - 2 WVCC 2012

Lomustine induced renal toxicity in dogs: a retrospective study with a prospective follow-up.

Spela Bavcar
(Spela Bavcar, Arno Roos, and Johan de Vos)

De Ottenhorst , Veterinary Oncology Referral Centre, Animal Hospital Zeeuws-Vlaanderen,

Terneuzen, Netherlands

[email protected]

Introduction
Lomustine induced renal toxicity is not well documented in dogs. In humans it is a late complication
after high cumulative doses. Our study had a retrospective arm, determining frequency and time of
occurrence of renal toxicity in tumour bearing dogs treated with lomustine. Subsequently, the
results were prospectively applied in new patients.

Materials
Inclusion criteria: lomustine delivered at least once with the patient alive &gt;21 days after the first
dose; creatinine (cut-off value 55+BW(kg) umol/l) determined before the first dose (unless the dog
was <7 years of age), during and/or after the final lomustine delivery; any other cause of renal
toxicity must be excluded. Urine analysis was optional in the retrospective arm, but obligatory for
the new patients.

Results
208 dogs were treated with lomustine between 1-10-1999 and 1-11-2011. 75 met the inclusion
criteria of which 17 (22, 7%) developed renal failure. The median number of lomustine deliveries
and the median cumulative dose before developing renal disease was 4, 8 (range 1-12) respectively
346 mg/m2 (range 74-630). When urine analysis was performed, transient renal glucosuria was the
initial sign of renal damage, before an increase in serum creatinine.

Conclusions
Lomustine induced renal toxicity seems to be a significant side effect in dogs. In contrast to
humans, renal failure can occur early in the course of treatment, starting with glucosuria indicating
a proximal renal tubulus damage. Blood ureum/creatinine determinations, and a urine analysis
should be performed previous to every lomustine delivery, and need to be continued after the last
lomustine treatment for at least one year.
 n d
2 WVCC 2012 - 15 –

PRELIMINARY STUDY ON A COMBINED DOXORUBICIN-DACARBAZINE CHEMOTHERAPEUTIC

PROTOCOL FOR THE ADJUVANT TREATMENT OF CANINE HEMANGIOSARCOMA

Riccardo Finotello
(Riccardo Finotello, Veronica Marchetti, Damiano Stefanello, Eric Zini, Giuliano Bettini, et al)

Department of Veterinary Clinics, School of Veterinary Medicine, University of Pisa, San Piero a
Grado, Pisa, Italy

[email protected]

Introduction
Canine hemangiosarcoma shows high metastatic potential and short OS. The gold standard consists
of surgery followed by doxorubicin-based chemotherapy. In this prospective study, we compared
adjuvant doxorubicin-cyclophosphamide (AC) with doxorubicin-dacarbazine (ADTIC) chemotherapy
to determine safety and efficacy.

Materials
Dogs with histologically-confirmed, surgically-removed and completely staged hemangiosarcoma
were enrolled. Tumour characteristic, chemotherapy protocol, toxicity, TTM and OS were analyzed.
Dogs were divided in Group 1 (AC) and in Group 2 (ADTIC).

Results
Sixteen dogs with biologically aggressive hemangiosarcoma were enrolled; 9 received AC (Group 1)
and 7 had ADTIC (Group 2). In Group1, 2 dogs had BM toxicity (grade 1 and 3) and 4 had GI toxicity
(grade 1-3). In Group 2, 5 dogs had BM toxicity (grade 2-4) and 2 had GI toxicity (grade 1-2). In
Group 1, 8/ 9 dogs developed metastases; TTM was 90 days. In Group 2, 4/7 dogs developed
metastases; TTM was 365 days. TTM increased in ADTIC group (p=0.094). In Group 1, 9/9 dogs died,
7 due to hemangiosarcoma; the mean OS was 90 days. In Group 2, 4/7 dogs died due to
hemangiosarcoma and 3 dogs were alive at 450, 465 and 585 days. Group 2 showed a longer mean
OS (P=0.066).

Conclusions
ADTIC group had a positive trend for TTM and OS compared with AC group. Cyclophosphamide and
dacarbazine are both alkylating agents; however, dacarbazine shows increased fractional dose
intensity, resulting in higher summation dose intensity of ADTIC compared with AC. Furthermore,
ADTIC was well tolerated requiring no dose reductions.
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- 16 - 2 WVCC 2012

Rectal lymphoma - a case series of twelve dogs

Nele Van den Steen

(Nele Van den Steen, Jennifer Stewart, Gareth Maglennon, Debs Flack, Jody Blyth Tancock, et al)

Animal Health Trust, Newmarket, United Kingdom

[email protected]

Introduction
Gastrointestinal lymphoma is associated with a poor prognosis, and is most often of a T-cell origin.
The purpose of this retrospective study was to present a series of dogs with rectal lymphoma,
which appear to have a better prognosis and have a different immunophenotype.

Materials
Searching the clinical and pathological database of the Animal Health Trust (1998-2006) and Queen
Mother Veterinary Hospital of Cambridge University (2000-2006) retrospectively, identified twelve
histologically confirmed cases of rectal lymphoma. Full staging was performed in 11/12 dogs.
Immunohistochemistry, with B- and T-cell markers (CD79a and CD3), was performed on eleven
samples.

Results
Six dogs underwent surgery and adjuvant chemotherapy with a median survival time of 936 days.
Three received only chemotherapy with a median survival time of 1347 days. Three of the dogs
who received chemotherapy were still alive and in remission at the time of censor after 1897, 1984
and 2664 days. One dog was euthanised six days after surgery for wound complications. One dog
did not receive any treatment and was euthanized 28 days after diagnosis. The final dog received
lactulose and prednisolone and was euthanized 172 days later with persistent dyschezia.
Immunohistochemistry was consistent with B-cell lymphoma in all analysed samples

Conclusions
Dogs with solitary rectal lymphoma, and particularly those treated with chemotherapy, seem to
have a considerably better prognosis than what has been reported historically for alimentary
lymphoma. Immunohistochemistry was consistent with a B-cell origin, in contrast to alimentary
lymphoma which is most often of T-cell origin.
 n d
2 WVCC 2012 - 17 –

PDGF receptors: new targeted therapy in canine osteosarcoma

Lorella Maniscalco
(Lorella Maniscalco, Selina Iussich, Emanuela Morello, Marina Martano, Fulvio Riondato, et al)

Dep of Animal Pathology, University of Turin, Italy

[email protected]

Introduction
PDGFR? and PDGFR? are tyrosine kinases receptors overexpressed in 70-80% of human
osteosarcoma (OSA) representing a suitable target for clinical use of specific kinases inhibitors.
Canine OSA is considered a model in comparative oncology. In this study we investigated PDGFRα
and PDGFRß expression in canine OSA and the in vitro effects of PDGFRs inhibition.

Materials
PDGFRα and PDGFRß expression was evaluated by immunohistochemistry on 28 cases of canine
OSA. Western blot and q-PCR specific for PDGFRs, PDGFs, p-AKT and p-MAPK were performed on
primary OSA cell lines. Apoptotic effect after treatment with specific PDGFR inhibitor (AG1296) was
evaluated by cytofluorimetry

Results
Immunohistochemistry revealed that canine PDGFRα and PDGFRß are expressed in 71, 4% and 82,
1% respectively. Western blot and q-PCR results showed that all canine OSA cell lines
overexpressed PDGFRα, PDGFRß if compared to normal osteoblastic cell line. Western blot analysis
showed that PDGFRs were able to activate AKT in all cell lines and MAPK in 5/7 cell lines. Moreover,
the in vitro treatment with AG1296 in “Penny” cell line induces apoptosis and down regulation of p-
AKT in a dose and time dependent manner.

Conclusions
These data showed that the expression and role of PDGFRs in canine osteosarcomas are similar to
human suggesting that PDGFRs could be considered new target for the treatment of canine
osteosarcoma and remarking the important role of canine model in testing innovative approaches
for human osteosarcomas therapies
 n d
- 18 - 2 WVCC 2012

The Utility of Staging in Canine Mast Cell Tumours

James Warland
(James Warland, Isabel Amores-Fuster, William Newbury, Malcolm Brearley and Jane Dobson)

The Queen’s Veterinary School Hospital, University of Cambridge, UK

[email protected]

Introduction
Although clinical stage is important for prognosis in canine mast cell tumours (MCT), authors
disagree on necessary investigations. These include local lymph node assessment, abdominal
ultrasound (±organ cytology), thoracic radiography, CBC/biochemistry, buffy coat analysis and bone
marrow aspiration/biopsy.

Materials
Retrospective analysis was undertaken of the presenting stage &amp; progression of dogs
presented to a referral hospital with MCT (1997-2008). Dogs included had confirmed MCT and
staging to include, at least, local lymph node assessment, abdominal ultrasound and thoracic
radiography (n=222).

Results
152 had no detectable metastasis; 68 had local lymph node metastasis and 15 had distant
metastasis (to liver, spleen, skin, regional lymph nodes). None had distant metastasis without local
node involvement. Two had thoracic metastatic nodules; both had another, more metastatic,
neoplasia. 217 were followed up (5 excluded - concurrent neoplasia). 30 animals followed for less
than 6 months were considered “lost to follow up” (LTFU). Of 149 dogs with no metastatic disease
at presentation, 23 were LTFU and 33 developed subsequent MCT disease; 15 developed
metastasis related to the original tumour, and 18 developed other MCT disease. Metastatic disease
was always detectable in the local lymph node including in those with distant metastases. All
subsequent metastases were to the skin, local/regional lymph nodes, liver or spleen; none were
pulmonary.

Conclusions
This study suggests that the local draining lymph node is sentinel to the staging of MCT. Abdominal
imaging is only necessary if this sentinel lymph node shows evidence of metastasis. Thoracic
radiographs are not a useful modality for staging MCT disease.
 n d
2 WVCC 2012 - 19 –

Anti-angiogenesis and increased mural cell coverage by low-dose cyclophosphamide with

piroxicam in canine malignant melanoma xenografts.

Nan Choisunirachon
(Nan Choisunirachon, Takayuki Nakagawa, Manabu Mochizuki and Nobuo Sasaki)

Laboratory of Veterinary Surgery, Department of Veterinary Medical Science, Graduate School of

Agricultural and Life Sciences, The University of Tokyo, Japan

[email protected]

Introduction
Thrombospondin-1 (TSP-1), induced by metronomic cyclophosphamide, causes endothelial
apoptosis. TSP-1 may assist vascular normalization by decreasing VEGF/VEGFR2 signaling, resulting
in an increase in mural cell coverage. The purpose of this study was to clarify the anti-angiogenic
effect of low-dose cyclophosphamide with piroxicam by measuring micro vessel density (MVD) and
vascular normalization in a canine melanoma xenograft model.

Materials
Nude mice xenografted with canine malignant melanoma cell line were randomly assigned to the
control (n=6) and treatment groups; low-dose cyclophosphamide (Cy group; n=6), piroxicam (PO, Pi
group; n=5), and combination of both treatments (Comb group; n=6) for 30 days and tumor growth
was observed. Harvested tumor tissues from nude mice were double immunostained with CD31
and alpha-SMA for mural cell marker. TSP-1 and VEGF expressions were determined by western
blot analysis.

Results
In the Comb and Cy groups, tumor volume was significantly smaller than in other groups (p<0.05).
Tissues of Comb group showed a significant decrease in MVD and an increase in mural cell-positive
vessels while those of Cy group revealed the lowest mural cell-positive vessels (p<0.05). TSP-1
expression in Cy group was lower than those of other groups. VEGF expression in Pi and Comb
groups was significantly lower than other groups (p<0.05).

Conclusions
Low-dose cyclophosphamide may decrease MVD due to endothelial cytotoxicity but increase
pathogenic vascular trait through decline in TSP-1 in this canine melanoma xenograft model.
Piroxicam combined with low-dose cyclophosphamide may improve vascular normalization through
anti-VEGF signaling.
 n d
- 20 - 2 WVCC 2012

Toxicity and Pharmacokinetic profile of a new anticancer agent in canine mammary cancer

Veronica Kristiansen
(Veronica Kristiansen, Sagita Dewi, Thora Jonasdottir and Stig Larsen)

Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, Oslo
Norway

[email protected]

Introduction
BP-C1 is a novel antitumor agent of low dose Cisplatinum combined with benzo-polycarbonic acid.
Good tolerability and antitumor effect has been shown in similar human study of metastatic breast
cancer patients. The aim of this study was to estimate the Maximum Tolerable Dose (MTD) and the
pharmacokinetic (PK) profile in dogs.

Materials
Two healthy dogs and nine with mammary cancer were planned to be included with five dogs
included by now. The study was performed as an open within patient 3-level Response Surface
Pathway design trial. Based on human data the cumulative dose-window was predefined as 0.07-
0.45 mg/kg resulting in an initial cumulative dose of 0.26 mg/kg. The dogs received one daily
intramuscular injection in three 7-days-periods. Platinum concentration was measured by blood
sampling 30 minutes after each injection and by serial sampling during 48 hours following first and
last injection in the first treatment period. Adverse effect was evaluated using VCOG-CTCAE criteria.

Results
All dogs went through all three dose-levels and reached the upper limit of the dose-window. MTD
was not reached. Adverse effects were rarely observed, and the maximum registered was grade 1.
The PK profile was found to follow a 2-compartment model with Tmax=30 minutes, t1/2?=18
minutes and t1/2?=98 hours.

Conclusions
BP-C1 was well tolerated. MTD was not reached within the dose-window used in this study. The PK
profile followed a 2-compartment model with a long release phase.
 n d
2 WVCC 2012 - 21 –

The use of adjuvant protocols with docetaxel and doxorubicin in cats with III grade mammary
cancer.

Ksenia Lisitskaya
(Marina Yakunina, Ksenia Lisitskaya and Alexander Shimshirt)

Clinic of Experimental Therapy ‘Biocontrol’ Russian Oncology Research Center, Moscow, Russian
Federation

[email protected]

Introduction
Mammary cancer is common in cats, and the postexcisional survival of affected cats is significantly
associated with tumor size or WHO clinical stage. Cats with advanced disease (stage III) have a poor
prognosis and decreased survival time. Previous studies suggested that systemic adjuvant
chemotherapy with doxorubicin improved the median survival time in cats with stage III disease.
The aim of the study was to determine the efficacy of a treatment with docetaxel in cats with stage
III mammary cancer.

Materials
65 cats with III grade mammary cancer that received prior mastectomy were included in the study.
Cats were divided in three groups: control group 1 (n=18), doxorubicin group 2 (n=36), doxetacel
group 3 (n=11). Doxorubicin (30 mg m?2) and doxetacel (Taxotere, 30 mg m?2) were administered
intravenously in 21-day cycles. All cats received two cycles of the proposed protocols. Response to
treatment was assessed as median time to progression (MTP) and median survival time (MST).

Results
In control group 1 MST was 4.15 mo, MTP was 4.05 mo, with only 5.3% of cats alive at 1 year. In
group 2 received doxorubicin-based chemotherapy MST was 8.7 mo, MTP was 8.3 mo, 1-year
survival rates were 41.7%. In group 3 received docetaxel-based chemotherapy MST was 11.7 mo,
MTP was 11.3 mo. In group 3 the survival rate at one year was 43%.

Conclusions
Advanced feline mammary cancer had a good response to systemic therapy with doxorubicin and
docetaxel, with no significant difference in median survival times.
 n d
- 22 - 2 WVCC 2012

Expression of oestrogen-α and progesterone receptors on Feline Endometrial Adenocarcinomas

Ana Laura Saraiva

(Ana Laura Saraiva, Alexandra Rêma, and Rita Payan-Carreira)

Escola Universiária Vasco da Gama, Coimbra, Portugal

[email protected]

Introduction
Endometrial adenocarcinomas are considered to be rare in domestic animals, with the exception of
cattle and rabbits. The low incidence of these tumours in cats may be related to their
underdiagnosis, since post mortem evaluation of female genital tract is not always performed and
because the submission of surgical specimens for morphological diagnose is still not common in
clinical practice. In our study, we pretend to achieve a more precise characterization of these
tumours, comparing them with normal endometrium

Materials
Fifty feline endometrial adenocarcinomas were identified by a minimum of three pathologists on
conventional haematoxylin and eosin-stained sections. We also selected samples of histologically
normal feline uterus (12 samples collected during progestagenic phase and 17 samples collected
during oestrogenic phase), used as controls. Immunolabelling was performed by the indirect avidin-
biotine-peroxidase immunohistochemistry method, using antibodies against ER-α (clone ER12; Cell
Marker®; 1:40) and PGR (clone NCL-L-PGR; Novocastra ®; 1:30).

Results
Feline endometrial adenocarcinomas under study were morphologically classified as papillary
serous carcinoma, clear cell carcinoma and “in situ” carcinoma. Concerning to the immunostaining,
control uterus were positive for both ER-α and PGR. All tumours lost ER-α expression, except clear
cell carcinomas, which maintained some degree of expression. PGR expression was preserved by all
neoplasms.

Conclusions
As far as we concerned, this is the first study performed on a large case series of feline endometrial
adenocarcinomas. Furthermore, morphological classification is performed by the first time. The
results concerning to ER-α and PGR expression confirm the resemblance of these lesions with those
described in women.
 n d
2 WVCC 2012 - 23 –

Classification of canine seminomas as classical or spermatocytic using morphology and

immunohistochemistry

Tor Espen Thorvaldsen

(Tor Espen Thorvaldsen, Ane Nødtvedt, Gjermund Gunnes and Tom Grotmol )

Norwegian School of Veterinary Science, Oslo, Norway

[email protected]

Introduction
Seminomas in the dog have traditionally been assumed to resemble human spermatocytic
seminomas, based on their low malignancy and the high age at which they occur. However,
recently published studies indicate that seminoma in the dog can be classified as classical and
spermatocytic seminomas in a similar way as in humans.(1) Furthermore, Grieco et al. (2007)
showed that classical seminoma comprises a substantial fraction of seminoma in the dog, thus
increasing its potential as a relevant model for human testicular cancer. The aim of the present
study was to investigate, by means of morphological, histopathological and immunohistochemical
evaluation, the occurrence of classical seminoma among Norwegian dogs.

Materials
By applying diagnostic criteria from human pathology, 45 seminomas from the Norwegian Canine
Cancer Register were examined histologically with Hematoxylin and Eosin (HE) and periodic acid-
Schiff (PAS) stains. All sections were stained immunohistochemically with antibodies against human
placental alkaline phosphatase (PLAP) and the transmembrane reseptor KIT (CD117).

Results
Although two of the seminomas showed immunohistochemical staining characteristics indicative of
classical seminoma (PLAP+/CD117+), all 45 examined seminomas were morphologically consistent
with spermatocytic seminoma.

Conclusions
Regional or breed differences in the occurrence of classical seminoma in the dog might explain the
discrepancy between the findings in the current study and the results presented by Grieco et al.
(2007). The differences warrant further investigation. 1.Grieco V, Riccardi E, Rondena M, Ciampi V,
Finazzi M. Classical and spermatocytic seminoma in the dog: histochemical and
immunohistochemical findings.

J Comp Pathol 2007 July;137(1):41-46.

 n d
- 24 - 2 WVCC 2012

Cancer stem cells from three ontogenetically distinct canine tumors have shared patterns of gene
expression

Aric Frantz
(Aric Frantz, Aaron L Sarver, Erin B Dickerson, Timothy D O’Brien and Jaime F Modiano)

University of Minnesot, Minneapolis United States

[email protected]

Introduction
Cancer stem cells (CSC) have been demonstrated in a variety of cancers; however, shared
properties have not been reported as a recurrent characteristic of these cells in different tumors.
We hypothesized that CSC from three ontogenetically distinct cancers share molecularly defined
properties.

Materials
CSC were enriched from canine hemangiosarcoma (HSA, N=4), osteosarcoma (OS, N=3), and
glioblastoma (GBM, N=4) by formation of multicellular tumor spheroids in serum-free conditions.
Genome-wide gene expression profiles were evaluated in these 11 sphere cell samples and in the
conventional culture monolayer cells from which each was derived using the Agilent microarray
platform.

Results
Sphere cells had traits of CSCs in vitro and in vivo, including increased resistance to cytotoxic drugs,
exclusion of vital dyes in the side population assay, differential expression of ABC transporters, and
increased capacity to engraft and survive as xenografts under conditions of limiting dilution. The
sphere cells had comparable and consistent changes in their genome-wide gene expression profiles
when compared to the monolayer cells, indicating enrichment of CSCs was among the parameters
driving molecular stratification of these tumors. However, other parameters also were discernible,
including highly tumor-specific properties.

Conclusions
We interpret the data to indicate CSCs arise through multiple independent mechanisms in solid
tumors, but a defined molecular pattern is present in cells with increased drug resistance and
tumor-initiating capacity. Ongoing work will define both the shared and unique molecular
properties of CSCs from these different cancers.
 n d
2 WVCC 2012 - 25 –

Role of Sulf1 and Sulf2 Splice Variants in Canine Mammary Tumour Growth

Lisa Pritchard
(Lisa Pritchard, Stephen Baines, Ken Smith and Gurtej Dhoot)

Royal Veterinary College, London, UK

[email protected]

Introduction
Extracellular sulfatase enzymes, Sulf1 and Sulf2, can alter tumour growth characteristics due to
their ability to regulate the activity of many growth factors and signaling molecules either positively
or negatively by removing the 6-0 sulfate group on heparan sulfate proteoglycans required for co-
receptor function. This role of Sulf1/Sulf2 in recent years, however, has become controversial due
to divergent changes observed in these isozymes in some tumours despite having similar functions
and substrate specificities. We hypothesised that some of these differences may relate to isoform
composition.

Materials
We examined the expression patterns of both Sulf1 and Sulf2 variants in a number of histologically
graded canine mammary tumours to determine if the association of certain variants with specific
cell signaling pathways worsened prognosis. Sulf1 and Sulf2 isoforms were identified using
antibodies to specific catalytic domain regions that would alter enzymatic activities and cell
signaling.

Results
Our data identified multiple variants of both Sulf1 and Sulf2 enzymes showing distinct patterns of
expression in hyperplastic mammary lesions versus tumours of high and low histological grade.

Conclusions
This study indicates that sulfatase expression occurs in both hyperplastic and neoplastic lesions of
the canine mammary gland and that there is variable expression of different isoforms. Further work
will involve association of different splice variants with ligands such as FGF and EGF that require 6-0
sulfation for signal progression and correlation of full length sulfatases with markers of the Wnt
signaling pathway.
 n d
- 26 - 2 WVCC 2012

Correlation between number of Gr1-positive myeloid precursor cells, p-STAT3 expression and
gene expression pattern in canine mammary cancer metastasis

Joanna Mucha
(Joanna Mucha, Magdalena Król, Olga Musielak, Kinga Majchrzak, Joanna Mucha et al)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences - WULS, Warsaw Poland

[email protected]

Introduction
The very recent studies on human and mice models have indicated an important role of myeloid
precursor cells in the tumor progression and metastasis. They are thought to suppress the immune
system and promote angiogenesis via Signal transducer and activator of transcription 3 (STAT3)
activation. As of now there is no data available on the correlation of Gr1-positive cell number,
phosphorylated STAT3 (p-STAT3) expression and cancer ability to metastasis.

Materials
50 metastatic and not-metastatic canine mammary tumors were subjected to
ummunohistochemistry to count number of myeloid precursor cell and p-STAT3 expression. Gene
expression using DNA microarrays was analyzed in metastatic canine mammary cell lines.

Results
We showed that the number of Gr1-positive cells and p-STAT3 expression are significantly higher
(p<0.001) in the metastatic tumors than in the non-metastatic ones. We also observed higher
expression of p-STAT3 in the canine mammary cancer cell lines with metastatic potential than in
other cell lines (p<0.001). Moreover, the number of myeloid precursors and p-STAT3 expression in
metastatic tumors correlate strongly. Microarray analysis of metastatic canine mammary cancer
cell lines revealed significant up-regulation genes involved in angiogenesis and interactions with
hematopoietic cells.

Conclusions
The tumor infiltrating myeloid precursor cells may invigorate the STAT3 activity (probably via
vascular endothelial growth factor – VEGF) that contributes to the tumor angiogenesis and
furthermore tumor`s ability to metastasize. The analysis of gene expression in canine mammary
cancer cell lines with metastatic potential indicated that semaphorin 3B (SEMA3B) and neuropilin
receptors (NRP) may also be important elements in this process.
 n d
2 WVCC 2012 - 27 –

Patterns of Ctip2 Immunoreactivity in Feline Oral Squamous Cell Carcinoma

Krystal Claybrook-Harris
(Krystal Claybrook-Harris, Howard Gelberg, Kay Fischer, Gitali Ganguli-Indra, Arup Indra, Mark Leid,
Stuart C Helfand)

Oregon State University, College of Veterinary Medicine, Corvallis, United States

[email protected]

Introduction
Ctip2 (COUP-TF interacting protein) is a transcriptional regulator present in normal human head
and neck epithelium and is important for cell proliferation. Ctip2 is upregulated in poorly
differentiated human head and neck squamous cell carcinoma and its expression is prognostic. We
hypothesized that differences in Ctip2 expression in feline oral squamous cell carcinomas (FOSCCs)
would also correlate with tumor differentiation. This study validated an immunohistochemical (IHC)
method for detection of Ctip2 in FOSCC and determined if its expression correlated with
histological features of malignancy.

Materials
Archived, formalin-fixed, paraffin-embedded FOSCC biopsies submitted to the Oregon State
University Veterinary Diagnostic Laboratory were used to prepare serial tissue sections. They were
heat treated in a commercial antigen retrieval solution, and immunostained with monoclonal
antibodies specific for Ctip2 , Ki67, and epidermal growth factor receptor (EGFR). Antibodies for
Ki67 and EGFR have previously been validated for the cat. Patterns of Ctip2 staining and tumor
differentiation were evaluated by a single pathologist (HG) using histological grading and IHC
scoring systems.

Results
Twenty FOSCC biopsies were examined. High grade tumors predominated. There were significantly
more Ctip2-positive cells in the high grade tumors. Tumors with a mitotic index >20 had widespread
Ctip2 positivity. Expression of EGFR and Ki67 scores did not correlate with Ctip2 expression or
cellular differentiation.

Conclusions
Expression of Ctip2 correlates with degree of tumor differentiation in FOSCC, which may be useful
for grading FOSCC. Its role in high grade FOSCC awaits clarification.
 n d
- 28 - 2 WVCC 2012

GENERAL ABSTRACTS
 n d
2 WVCC 2012 - 29 –

Winner of the Basic Science Award of the Dutch Animal Cancer Foundation

Expression of Sfrp1 and activation of the Wnt-pathway in healthy and affected adrenal glands of
ferrets

Marja K. de Jong
(Marja K de Jong, Nico J Schoemaker and Jan A Mol)

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht

University, Utrecht, The Nederlands

[email protected]

Introduction
Gonadectomy induced adrenocortical tumors in ferrets and mice are thought to share a similar
pathogenesis. In mice, the tumor suppressor gene Secreted Frizzled Related Protein 1 (Sfrp1), a
dominant inhibitor of the Wnt-pathway, has been proposed as a candidate gene for initiation of
these tumors. Whether Sfrp1 and the Wnt-pathway play a similar role in the oncogenesis of
hyperadrenocorticism in ferrets is investigated in this study.

Materials
The expression of Sfrp1 and three target genes within the Wnt-pathway (cMyc, Axin2 and CyclinD1)
were studied in 14 adrenal glands from healthy ferrets and in 14 adrenocortical tumors by means of
quantitative polymerase chain reactions. Nuclear β-catenin, a reliable indicator of Wnt-pathway
activation, was assessed.by immunohistochemistry.

Results
Contrary to what was expected, results demonstrate that Sfrp1 is up regulated and Axin2 is down
regulated in the tumor group compared to the control group. Nuclear β-catenin staining confirms
the down regulation of the Wnt-pathway in adrenocortical tumors in ferrets.

Conclusions
The findings suggest that it is unlikely that the role of Sfrp1 in the pathogenesis of adrenocortical
tumors in ferrets is similar to its proposed role in mice. Remarkably, there are several studies
concerning other types of gonadotropin dependent tumors which show findings similar to this
study.
 n d
- 30 - 2 WVCC 2012

TOTAL CYSTECTOMY AND URETERO- URETHRAL/PREPUTIAL/VAGINAL ANASTOMOSIS IN 23 DOGS

WITH TRANSITIONAL CELL CARCINOMA OF BLADDER

Tsuyoshi Kadosawa
(Tsuyoshi Kadosawa, Ai Watabe, Masao Yamashita, Makiko Tominaga, Yosifumi Endo, Kazuko
Hirayama and Hiroyuki Taniyama,)

Dept. of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University
Ebetsu Japan

[email protected]

Introduction
The trigonal or urethral involvement of canine bladder transitional cell carcinoma (TCC) often cause
fatal urinary tract obstruction. The purpose of this study was to evaluate total cystectomy and
uretero- urethral/preputial/vaginal anastomosis as a potential curative procedure for dogs with
such aggressive bladder TCC.

Materials
Inclusion criteria were dogs treated with curative intent cystectomy with uretero-
urethral/preputial/vaginal anastomosis. All dogs had locally aggressive TCC with multifocal lesions,
trigonal or urethral involvement. Any dogs with nodal or distant metastases at presentation were
excluded.

Results
Ten male dogs and 13 female dogs were included. The bladder was removed in all dogs, including
the prostatic urethra in 2 dogs and the total urethra in 13 dogs. The ureters were anastomosed to
urethra in 8 dogs, to prepuce in 7 dogs and to vagina in 8 dogs. Regional lymph nodes were
removed in the latest 11 dogs, and 3 dogs had involved ones. Surgical margins were completed in
19 dogs, and incompleted in 4 dogs with the ureteral or prostatic urethral involvement. No local
recurrence was observed, but nodal or distant metastasis developed in 9 dogs. The overall median
survival was 11 months. Three dogs are still alive. A diaper was used for urinary incontinence in all
dogs. All owners were satisfied with their dogs’ quality of life.

Conclusions
Although the removal of the bladder will cause urinary incontinence, the postoperative care was
manageable for all owners. A total cystectomy and uretero- urethral/preputial/vaginal anastomosis
should be considered for dogs with locally aggressive TCC.
 n d
2 WVCC 2012 - 31 –

Electroporation enhances bleomycin efficacy in cats with advanced head and neck carcinoma

Enrico Pierluigi Spugnini

SAFU Department, Regina Elena Cancer Institute of Rome, Italy

[email protected]

Introduction
Advanced head and neck carcinoma represents a significant health in cats. Electrochemotherapy
(ECT) is a tumor treatment that combines the systemic or local delivery of anticancer drugs with the
application of permeabilizing electric pulses. In this study we evaluate the capability of ECT to
improve bleomycin efficacy in cats with advanced head and neck carcinoma.

Materials
A cohort of eleven cats with inoperable tumors were treated with intravenous bleomycin (15
mg/m2) coupled with ECT under sedation. Further weekly sessions of ECT (2 to 8) were performed
until complete response or tumor regrowth occurred. More precisely, five minutes after the
injection of the chemotherapy agent, sequences of 8 biphasic pulses lasting 50+50 microseconds
each, with 1 ms interpulse intervals, were delivered in bursts of 1300 V/cm using caliper electrodes.
Adherence of the external electrodes to the lesion was maximized using an electroconductive gel.
Their responses were matched to those of ten controls treated with bleomycin alone.

Results
The toxicities were minimal and mostly treated symptomatically. ECT resulted in increased local
control with a 90% of complete responders while the controls had a response rate of 30%. Median
time to recurrence in the ECT group was 60 weeks, while in controls it was 2 weeks (p

Conclusions
We conclude that ECT is a safe and efficacious therapy for head and neck carcinoma in cats; its use
may be considered as part of strategies for the reintroduction of drugs with a narrow therapeutic
index in the clinical protocols.
 n d
- 32 - 2 WVCC 2012

Radioactive microbrachytherapy for head and neck tumors in dogs and cats

Jolle Kirpensteijn
(Jolle Kirpensteijn, Sebastiaan A van Nimwegen and Frank Nijsen)

Utrecht University, Utrecht, Netherlands

[email protected]

Introduction
Head and neck cancers are common in companion animals. A potentially effective treatment option
for patients with head and neck malignancies of limited size for whom no treatment options are
available, could consist of directly intratumorally injected microspheres loaded with the combined
beta-gamma emitting radioisotope holmium-166 (166Ho).

Materials
Microspheres were administered directly into the tumors of 8 veterinary patients with spontaneous
head and neck tumors; 5 cats with an oral squamous cell carcinoma and 3 dogs with a squamous
cell carcinoma in tongue and neck (n=1 and n=1, respectively) and one with a fibrosarcoma in the
mandible were treated by intratumoral injections with 166Ho microspheres.

Results
The injections of the radioactive microspheres were technically successfully performed. Generally,
the treatment was well tolerated by the animals and the clinical condition of 6/8 animals improved
markedly. In the majority of patients, partial response was achieved, which frequently led to
downstaging to surgical resection. However in three of the cats with oral squamous cell carcinoma,
outcome was complete remission. In only two animals progressive disease was observed. Toxicity
was absent in most animals. In one patient a skin defect was observed. Most biochemical and
hematological parameters normalized after treatment. The life of the majority of animals was
extended whilst associated with a good quality of life.

Conclusions
For the small lesions (1-15g; n=6), treatment often was curative whereas treatment of large lesions
(95-150g) resulted in downstaging to surgical resection.
 n d
2 WVCC 2012 - 33 –

ACCELERATED HYPOFRACTIONATED RADIATION THERAPY FOR THE TREATMENT OF FELINE ORAL

SQUAMOUS CELL CARCINOMA: A PILOT STUDY

Valerie J Poirier
(Valerie J Poirier, Rodney C Straw, Barbara Kaser-Hotz and David M Vail)

Australian Animal Cancer Fundation/Brisbane Veterinary Specialist Centre Albany Creek Australia

[email protected]

Introduction
Squamous cell carcinoma (SCC) is the most common feline oral tumor. Radiation therapy and/or
surgery are the standard of care in humans and dogs. In the cat, standard radiation protocols with
curative intent or palliative intent only achieve tumor control of 1.5 to 5.5 months. The purpose of
this study was to evaluate progression free survival (PFS), local progression free survival (LPFS) and
overall survival (OS) for feline oral SCC treated with an accelerated hypofractionated radiation
therapy (RT).

Materials
Cats with histologically confirmed oral SCC and T1-3N0M0 were treated with 10 daily fractions of
4.8 Gy.

Results
Seventeen cats with oral SCC underwent RT. Thirteen had macroscopic disease and 4 were treated
in the microscopic setting. All cats completed the prescribed radiation protocol. All cats had
concurrent esophageal tube placement and acute effects were manageable and self-limiting.
Response could be assessed in 13 patients (5 CR and 7 PR). Four cats (23%) developed metastatic
disease without evidence of local progression. The median PFS was 123 days (with a 1 year PFS of
25%), the medial LPFS was 323 days (with a 1 year LPFS of 48%) and the overall median survival was
235 days (with a 1 year OS of 34%).

Conclusions
This accelerated hypofractionated RT protocol was generally well tolerated with acceptable adverse
events. Tumor response was observed and long term tumor control was achieved in some cats.
 n d
- 34 - 2 WVCC 2012

Response Rate and Duration Associated with a 4Gy 5 Fraction Palliative Radiation Protocol

Catie McDonald
(Catie McDonald, Catie McDonald and Jayme Looper)

VCA Raleigh Hills Animal Hospital Beaverton, United States

[email protected]

Introduction
The purpose of this retrospective study was to determine if 4Gy fractions over 5 consecutive days is
an effective and safe palliative radiation protocol for dogs and cats.

Materials
Eighty patients (22 cats, 58 dogs) with complete follow up information were evaluated.

Results
Overall response rate (ORR) for all patients was 67%. Median progression free survival (MPFS) was
3.3 months and median survival (MST) was 4.2 months. Primary bone tumors were the most
common tumors treated. The ORR for primary bone tumors was 66.6%, the MPFS was 3.5 months
and MST was 3 months. The most common tumor treated in cats was oral squamous cell carcinoma
and ORR was 54.5 %, the MPFS was 1.8 months and MST was 3 months. Soft tissue sarcomas were
the second most common tumor treated in dogs (10). ORR was 80% and the 2 other patients had
stable disease. MPFS was 5.7 months and MST was 7.9 months. Overall rate of toxicity was 18.4% in
65 sites that were evaluated for toxicity. Acute toxicities were all grade I or II and occurred in 16.9
% of patients evaluated. All late toxicity was grade I alopecia and leukotrichia.

Conclusions
There appears to be a comparable response rate for this palliative protocol as compared to others
historically. This response was seen over a wide range of tumors. We also documented a low
toxicity profile in a shorter overall treatment time, making this protocol more attractive for some
clients.
 n d
2 WVCC 2012 - 35 –

Phase I Clinical Trial of Intratumorally Administered Radioactive Gold Nanoparticles in Canine

Prostatic Carcinoma

Sandra M Axiak
(Sandra M Axiak, Jimmy Lattimer, Kim Selting, Carolyn Henry, Jeff Bryan, et al)

University of Missouri, Columbia, United States

[email protected]

Introduction
Gum Arabic coated radioactive gold nanoparticles (GA-198AuNP) have shown effective diffusion,
retention, and ~80% reduction in tumor volume in the mouse model of human prostatic carcinoma.
Our objective was to determine the toxicities associated with GA-198AuNP therapy when
administered to dogs with spontaneously occurring carcinoma of the prostate.

Materials
Dogs diagnosed with carcinoma of the prostate by cytology or histopathology, and with no
evidence of bladder invasion, were eligible for enrollment. Injection of GA-198AuNP was achieved
with CT guidance. A complete blood count, chemistry panel, and urinalysis were performed prior to
treatment and then weekly for 4 weeks following treatment; a CT scan was performed 3-4 weeks
following treatment.

Results
Seven dogs have been enrolled to date. Two dogs were treate to a biologically equivalent dose of
50Gy, and 5 dogs were treated to a biologically equivalent dose of 105 Gy. Six of the 7 dogs
completed the 4 week trial period, and no evidence of local or systemic toxicity was noted in these
dogs. One dog died due to bladder rupture 10 days following treatment. Necropsy was not
performed on this dog, and bladder rupture which could have resulted from tumor progression or
swelling/ abcessation.

Conclusions
Intratumoral administration of GA-198AuNP caused no acute systemic toxicities in any dog,
however, local tumor swelling is possible and dogs must be closely monitored for evidence of
urethral obstruction. Trials investigating efficacy of this treatment are warranted and ongoing.
 n d
- 36 - 2 WVCC 2012

Current Clinical Practice in Target Volume and Dose Specification Reporting in Veterinary
Radiation Oncology

Michelle Turek
(Michelle Turek, Jessica Lawrence and Brittany Feldhaeusser)

University of Georgia College of Veterinary Medicine, Athens, United States

[email protected]

Introduction
Radiotherapy treatment outcomes can only be meaningfully interpreted with precise knowledge of
delivered dose. Due to irradiation technique limitations, variation in radiation dose occurs across
the target volume, and prescribed dose may not equal delivered dose. Non-standardized reporting
of dose and volume specifications may lead to false interpretation of a study and to erroneous
application of reported results. Unambiguous parameters must be used for reporting to ensure a
common language exists between treatment centers.

Materials
Clinical radiotherapy articles published between 2001 and 2011 were reviewed for patterns of dose
reporting. Only publications of externally-applied, non-IMRT, MeV photon radiotherapy in small
animals were considered. Details of volumes, margins, organs at risk and dose
specifications/variations were collected. Journal, practice-type, practice location and radiation-
oncologist participation were correlated with reporting practices.

Results
None of the 153 articles reviewed fully adhered to the current target volume and dose specification
recommendations of the International Commission on Radiation Units and Measurements (ICRU).
The most consistently reported parameter was prescribed dose (100%). Target volumes were
defined according to ICRU recommendations in <15% of publications, treatment margins were
defined in 50%, and <15% commented on exposure of organs at risk. A dose reference point in the
target volume was specified in <15% of articles. Dose and target volume specification were deemed
reproducible in less than half of reports.

Conclusions
Diversity exists in reporting practices used in the veterinary radiation oncology literature.
Unequivocal procedures for target volume and dose delivery specification are essential to permit
the relevant exchange of information between radiation facilities.
 n d
2 WVCC 2012 - 37 –

INVESTIGATION OF MASITINIB ON RADIOSENSITIVITY OF FELINE INJECTION SITE SARCOMA CELLS

Michelle Turek
(Michelle Turek, Jessica Lawrence, Corey Saba, Brittany Brouillire, Amanda Vance, et al)

University of Georgia Athens United States

[email protected]

Introduction
Platelet-derived growth factor receptor (PDGFR) has been shown to influence growth and viability
of injection-site sarcoma (ISS) cells. Masitinib mesylate is a potent tyrosine kinase inhibitor that
selectively targets PDGFR. Data are lacking regarding whether inhibition of PDGFR affects the
response of ISS cells to radiation. The ability to disrupt PDGFR signaling may represent a potential
therapy that sensitizes cells to radiation. The objective of this study was to determine if masitinib
alters radiosensitivity of ISS cells.

Materials
ISS cells in monolayer culture were plated and exposed to masitinib and a single dose of radiation.
Alamar Blue proliferation assays were used to generate dose-response curves following treatment
with increasing doses of masitinib (0-86 µM) and radiation (0, 3, or 6 Gy). Clonogenic assays were
used to generate cell survival curves following treatment with masitinib (0, 1µM) and radiation (0,
3, 6, or 9 Gy). DNA double strand break induction was measured via ELISA assay.

Results
Masitinib and radiation positively interacted with one another to inhibit cell proliferation.
Masitinib-treated cells displayed a similar dose-dependent inhibitory effect across various radiation
doses. Prolonged exposure to masitinib induced cell inhibition at doses

Conclusions
Preliminary data suggest that pretreatment with masitinib appears to enhance sensitization of ISS
cells to radiation and additional investigation is warranted.
 n d
- 38 - 2 WVCC 2012

Canine Glioma Cancer Stem Cells have Global Defects in DNA Damage Pathways induced by
Ionising Radiation

Lisa Pang
(Lisa Pang and David J Argyle)

Roslin Institute / Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom

[email protected]

Introduction
Gliomblastoma multiforme (GMB) is an aggressive and locally invasive primary brain tumour. In
dogs, GMB is a relatively common tumour accounting for 12% of brain tumours. Gliomas occur
most commonly in brachycephalic breeds, such as the Boston terriers and Boxers. Conventional
therapy has had limited efficacy. We have previously reported the identification and
characterization of canine glioma stem cells. In this comparative study between glioma stem cells
and parental cells, we aimed to characterise global gene expression differences in canonical DNA
damage pathways induced by ionizing radiation.

Materials
Putative cancer stem cells (CSCs) were isolated by tumoursphere formation from the canine GBM
cell line J3T, and further characterised by marker expression, sensitivity to doxorubicin and ionizing
radiation (IR), and invasiveness. Cells were treated with 5 Gy IR and harvested 4 hours post-
treatment. RNA was isolated and hybridized to the Affymetrix Canine_2.0 gene chip. Initial data
analysis was done in Partek and Ingenuity Pathway Analysis was used for canonical pathway
analysis.

Results
Here we report that CSCs can be isolated form a canine glioma cell line, J3T. We show that these
cells are resistant to doxorubicin and IR. Utilising the Affymetrix canine microarray, we are able to
definitively show that there are significant differences in global gene expression profiles, and IR-
induced DNA damage pathways, of isolated the cancer stem cells and the parental cells.

Conclusions
Extensive analysis of defects in DNA damage induced pathways of cancer stem cells will enable the
development of therapies to target the elusive cancer stem cell population.
 n d
2 WVCC 2012 - 39 –

Src Blockade Mediates Potent Inhibitory Effects on Growth and Survival of Canine
Hemangiosarcoma

Stuart C. Helfand
(Stuart C. Helfand, Kevin Marley, Jeff Tyner, Marc Loriaux and Brian Druker)

Oregon State University, Corvallis, United States

[email protected]

Introduction
Canine hemangiosarcoma (HSA) remains one of the most intractable malignancies in veterinary
oncology. Recent studies indicate that HSA cells utilize tyrosine kinase signaling cascades to sustain
growth, providing an opportunity for therapeutic intervention. We previously screened a canine
HSA cell line against 60 tyrosine kinase inhibitors and Src inhibition appeared to exert an
antiproliferative effect. In this study, we used the Src inhibitor, dasatinib, to investigate its effects
on HSA signaling and proliferation, and compared responses to those of imatinib, a kit, bcr-abl,
PDGFR-ß inhibitor. The combined effect of added doxorubicin was also evaluated.

Materials
We used three validated canine HSA cell lines, SB-HSA, Den-HSA, and OR-HSA. Cells were incubated
in 72 hr cytotoxicity assays using an MTS readout. Cells were cultured with and without dasatinib or
imatinib at varying concentrations in triplicate and the means of replicates used for analysis.
Varying concentrations of doxorubicin were added alone and in combinations in some cultures.
Western blotting revealed effects of inhibitors on phosphorylation of Src and Erk 1/2.

Results
Dasatinib was 10-fold more potent than imatinib in mediating cytotoxicity, signal blockade, and
chemosensitization in all cell lines. Dasatinib was effective in nanomolar concentrations compared
to imatinib that required micromolar doses to obtain comparable effects. Dasatinib inhibited Src
tyrosine phosphorylation at 10 nM, which in turn inhibited phospho-Erk 1/2 downstream.

Conclusions
Blockade of Src signaling by dasatinib markedly suppresses HSA cell growth. The enhanced
cytotoxic effect of doxorubicin combined with dasatinib may help to inform future clinical
investigations.
 n d
- 40 - 2 WVCC 2012

Development of recombinant canine IgE Fc-based immunotherapy for canine mast cell tumours.

Richard Elders
(Richard Elders, Stephen Baines, Angela Holder and Brian Catchpole)

Animal Health Trust, Newmarket, United Kingdom

[email protected]

Introduction
Expression of FcεRIα,the high affinity receptor for IgE, is restricted to tissue mast cells and
circulating basophils, making it a potential therapeutic target. The hypothesis of these studies was
that recombinant canine (rc) IgE Fc constructs could be generated to target FcεRIα -expressing
canine mast cells.

Materials
Canine IgE and TRAIL (TNFSF10) sequences were identified in the Dog Genome Assembly. Selected
regions of the coding sequences were amplified by PCR from genomic DNA, cloned into a
mammalian expression vector (pSecTagA) and transfected into mammalian cells. Recombinant
proteins were characterised by Western blotting and ELISA, and their ability to bind to FcεRIα and
the C2 mastocytoma cell line investigated by ELISA and flow cytometry, respectively.

Results
The Fc portion of canine IgE (rcIgECH2-4), rcTRAIL169-282, and a fusion protein of the two were
successfully expressed. Western blotting demonstrated tagged proteins of the correct sizes.
Exploiting their tags for capture in an ELISA, the recombinant proteins were recognised by anti-IgE
and anti-TRAIL antibodies. Using the Allercept® system, the rcIgE Fc-containing proteins were
shown to bind to rFcεRIα, which could be inhibited by commercially available monoclonal IgE. Flow
cytometry demonstrated binding of rclgE Fc-containing proteins to C2 cells that express FcεRIα,
which could be inhibited by monoclonal IgE. No binding to cells lacking FcεRIα could be
demonstrated.

Conclusions
Recombinant canine IgE Fc, and a TRAIL fusion protein thereof, can bind to mast cells. This system
offers a platform for development of mast cell-targeted immunotherapy.
 n d
2 WVCC 2012 - 41 –

Transgeic models reveal delayed development and oncogenic effects in mouse mammary glands
overexpressing Runx2

Joanna Morris
(Joanna Morris, Alison Lindsay, Ewan Cameron, Laura McDonald, Anne Terry and Karen Blyth)

University of Glasgow, Glasgow, United Kingdom

[email protected]

Introduction
RUNX2 is a member of the RUNX transcription factor family which binds DNA to both activate and
repress gene transcription in various developmental processes and in cancer. Paradoxically Runx
genes may function as both oncogenes and tumour suppressor genes depending on cell context.
Recent evidence in human breast carcinoma cell lines suggests an oncogenic and metastatic role for
RUNX2 since inhibition of RUNX2 reduces invasiveness in vitro and tumorigenicity in an orthotopic
model.

Materials
To investigate the effects of ectopic Runx2 expression in vivo, transgenic mice were generated with
Runx2 targeted to mammary epithelium using the MMTV promoter. To test the oncogenic effect of
Runx2 these MMTV-Runx2 mice were crossed with two tumour prone mammary models (PyMT and
erbB2) and RUNX expression was examined immunohistochemically in gross mammary tumours of
mice, dogs and humans

Results
Runx2 overexpression caused a ductal branching and elongation defect during development of
virgin pubertal glands, and a lactation defect following parturition due to delayed differentiation of
alveolar cells during pregnancy. Mammary glands of aged MMTV-Runx2 females (>16 months)
displayed hyperplastic and preneoplastic changes, however Runx2 did not accelerate oncogenesis
in either the PyMT or erbB2 mammary tumour prone models. Variable RUNX2 expression was
detected in murine, canine and human breast tumours.

Conclusions
Runx2 overexpression produces a phenotype of delayed pubertal development, delayed alveolar
differentiation in pregnancy and hence impaired lactation. The mammary tumour prone models do
not support a direct oncogenic role for Runx2.
 n d
- 42 - 2 WVCC 2012

Epithelial to Mesenchymal Transition in Canine Breast Cancer

Alejandro Cervantes Arias

(Alejandro Cervantes Arias, David Argyle and Lisa Pang)

Roslin Institute / Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom

[email protected]

Introduction
Breast cancer in dogs affects 25% of unspayed bitches and approximately 50% of them are
malignant, making it one of the most important causes of death. The Epithelial-Mesenchymal
Transition (EMT) process is involved in embryogenesis, fibrosis, carcinogenesis, metastasis and
tumour recurrence. The Transforming Growth Factor-Beta (TGF-ß) pathway and its transcription
factors are crucial for EMT induction, during which epithelial markers are downregulated and
mesenchymal markers upregulated. Cells undergoing EMT become invasive, and acquire stem cell
characteristics, which may be relevant for metastasis and tumour recurrence and its targeting for
diagnosis and treatment.

Materials
Canine breast cancer cells were grown in Dulbecco’s modified Eagle’s medium. EMT was induced
through incubation with 10 ng/ml of TGF-β over a 6 day period, and our findings were confirmed by
cell morphology changes, western blot analysis, colony formation assay, in vitro wound healing
assay, and sphere forming ability.

Results
Cells treated with TGF-β showed mesenchymal characteristics and an increase in mesenchymal
markers and transcriptional factors with decreased epithelial markers. TGF-β treated cells
demonstrated greater ability to invade in wound healing assays, colonizing the wound faster.
Mammospheres (which are representative of cancer stem cells) showed upregulation of
mesenchymal markers, suggesting a link between EMT and cancer stem cells.

Conclusions
Collectively, the data confirmed that canine breast cancer cells undergo EMT. Further, cells that
undergo EMT have cancer stem cell characteristics, suggesting that there is a close link between
EMT and the stem cell phenotype during tumour cell migration and metastasis.
 n d
2 WVCC 2012 - 43 –

Outcome of triple negative mammary carcinomas (TNMC) in dogs.

Catherine Ibisch
(Catherine Ibisch, Frederique Nguyen, Floriane Morio, Laetitia Jaillardon, Tiffanie Godard et al)

Oniris, Nantes-Atlantic College for veterinary medicine, food science and engineering, Nantes,
France

[email protected]

Introduction
Five types of human breast cancer, identified by immunohistochemistry (IHC), have distinct
prognoses and therapeutic options. Recent studies indicate that canine mammary carcinomas
(CMC) resemble human breast cancers. We have demonstrated that the current molecular
classification of human breast cancer (Nielsen et al. 2004) could be of value in dogs and that the
most frequent subtype among canine invasive carcinomas was the “triple negative” (negative for
Oestrogen and Progesterone Receptors (ER, PR) and Her-2). We analyse here the outcome of dogs
bearing TNMC.

Materials
Clinical data including breed, neutering, age and weight at diagnosis, presence of metastases, cause
and time of death, were recorded for 275 invasive CMCs treated by surgery alone. Histological
records included the subtype of carcinoma (WHO 1999), Elston &amp; Ellis grade, presence of
emboli, lymph node metastasis and IHC stainings using ER, PR, Her2 (scored according to Wolff et
al.2007), CK5/6, EGF-R and Ki67. Analyses were independently reviewed by four pathologists.

Results
Among the TNMC, 56 % were basal-like (EGFR and/or CK5/6 +) and 20 % non basal-like. Histological
subtype was predictive for specific survival (p=0.001) as well as ER positive staining (p=0.046).
TNMC showed a significantly shorter specific survival (median=224 days) when compared to
luminal A CMCs (median=641 days) (p=0.016).

Conclusions
TNMC in dogs represent a particular clinical entity associated with shorter survival as reported in
humans. CMCs are an interesting model for breast cancer research, especially for the triple-
negative subtype for which efficient therapies are lacking.
 n d
- 44 - 2 WVCC 2012

Establishment of a new canine prostate carcinoma cell line with complex karyotype changes
involving polysomy 13

Saskia Willenbrock
(Saskia Willenbrock, Nina Eberle, Ingo Nolte, Hugo Murua Escobar, Nicola Reimann-Berg, Jörn
Bullerdiek,)

Small Animal Clinic and Research Cluster REBIRTH, University of Veterinary Medicine Hannover,
Hannover, Germany

[email protected]

Introduction
Besides man, the dog is the only animal developing spontaneous prostate cancer with significant
incidence showing a comparable disease progression and histopathology. Although prostate cancer
is of great importance in veterinary medicine as well as in comparative medicine, only few canine
cell lines are available. Herein, we report of the establishment of the new canine prostate cell line
DT08/40 and its cytogenetic characterization in comparison to the primary tumour material.

Materials
A prostate carcinoma fine-needle aspirate was transferred into sterile flasks containing 5 ml
medium 199. The cultures were incubated at standard conditions and subcultivated at confluency
twice a week. Colcemid [0.1 µg/ml] was added 2 h before chromosome preparation using routine
methods. Cell suspensions were dropped onto ice-cold slides, incubated for 7 days at 37°C followed
by GTG-banding according to a protocol previously described. Results were processed and recorded
with BandView 6.0 (Multi-Species). Karyotype description followed the nomenclature proposed in
our laboratory.

Results
The cells of DT08/40 grew very well and showed evidence of being spontaneously immortalized
when crossing the 20th passage with no changes in morphology and growth pattern in comparison
to the primary material. DT08/40 has been maintained in continuous culture actually exceeding 200
passages. Outcome of the cytogenetic analyses resulted in a polysomy 13 and complex karyotype
changes in the primary material as well as in DT08/40.

Conclusions
The cytogenetic analysis of DT08/40 revealed conserved chromosomal changes in comparison to
the primary material making this newly established cell line a valuable tool for prostate cancer
research.
 n d
2 WVCC 2012 - 45 –

Evidence for a causal association of ECPV-2 with equine squamous cell carcinoma

Sabine Brandt
(Sabine Brandt, Lisa Samek, Katharina Schönthaler and Sabine Sykora)

Equine Research Group, Equine Clinic, University of Veterinary Medicine, Vienna, Austria

[email protected]

Introduction
Despite the pronounced impact of squamous cell carcinoma (SCC) on the health and welfare of
affected horses, its aetiology is still unclear. The demonstrated association of human
papillomaviruses (HPV) with human SCC suggests that equine SCC may also be caused by PVs.
Recently we have identified a novel equine papillomavirus termed EcPV-2, which revealed features
that are typical for carcinogenic HPV types.

Materials
Using (RT-)PCR, we screened a series of samples derived from SCC-affected and healthy horses for
the presence of EcPV-2 DNA and mRNA.

Results
EcPV-2 DNA was detected in 23/25 equine genital SCCs/precursor lesions and 2/3 PBMC samples,
but not in normal skin surrounding the lesions. No viral DNA was traceable in 10 ocular SCCs
affecting the nictitating membrane. However, 5/5 ocular SCC metastases and one carcinoma in situ
involving the eyelid scored positive. PCR from 37 smegma samples and 54 vulval swabs from
healthy horses demonstrated EcPV-2 DNA in 2/37 and 2/54 samples. Clinical re-examination
revealed vulval SCC precursor lesions in one mare. In addition, information was obtained that 1/2
EcPV-2 positive geldings is co-stabled with a horse suffering from advanced penile SCC. Remarkably,
reverse transcription (RT)PCR carried out from tumour and PBMC RNA derived from severely SCC-
affected patients also demonstrated the presence of E6 and E7 mRNA, thus indicating that viral
genes are also transcribed.

Conclusions
Together with today’s knowledge on PV infection in humans and mammals, our recent findings are
indicative for an aetiologic association of EcPV-2 infection with equine SCC.
 n d
- 46 - 2 WVCC 2012

The effect of surgical stress on regulatory T cells in dogs with cancer

Ai Watabe
(Ai Watabe, Hiromichi Uesawa, Takamitsu Kimura, Dah-renn Fu, Katsuro Hagiwara, Yoshifumi Endo,
Tsuyoshi Kadosawa and Yosifumi Hagiwara)

Dept. of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University
Ebetsu Japan

[email protected]

Introduction
Surgical removal of the primary tumor provides the chance of recovery from immune suppression
induced by tumor. But surgery is invasive for patients and the following local cytokine storm has a
significant impact on immune status. In this study, we examined the changes in peripheral
lymphocyte subsets including regulatory T cells of dogs received definitive surgery for cancer.

Materials
To evaluate perioperative changes in the immune status of dogs, the immunophenotypes (CD3+,
CD4+, CD8+, CD21+ and CD4+FoxP3+) of peripheral blood lymphocytes and plasma concentration
of TGF-ß1 were measured in 44 various tumor-bearing dogs. The blood samples were collected on
the day of operation, 2nd, 2weeks and 1 month after operation.

Results
The relative percentage of regulatory T cells (CD4+FoxP3+) and WBC increased on the 2nd day and
decreased on the day 2 weeks after operation. Serum concentration of TGF-ß1 increased on the day
2 weeks after operation. The relative percentage of CD3+ lymphocyte increased slowly and CD4+,
CD8+ and CD21+ lymphocyte have no statistical changes.

Conclusions
Local surgical injury induced systemic changes, including raise of the relative percentage of
regulatory T cell and plasma concentration of TGF-ß1. These changes may lead to immune
suppression and growth or adhesion of tumor cells. Though surgery remains the appropriate and
necessary therapeutic modality for most patients with tumor and provides the chance of recovery
from immune suppression induced by tumor, new perioperative adjuvant therapies are necessary
for preventing immune suppression or improving immune status.
 n d
2 WVCC 2012 - 47 –

The effect of anti-RANKL antibody on canine osteosarcoma in a xenograft mouse model

Ayako Kamida
(Ayako Kamida, Takayuki Nakagawa, Tassanee Jaroensong, Manabu Mochizuki, Nobuo Sasaki, Jun
Hirose, Sakae Tanaka and Ryohei Nishimura)

Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Japan

[email protected]

Introduction
Canine osteosarcoma (COSA) is a common primary bone tumor with severe osteolysis, for which
osteoclasts (OCs) may play an important role. OCs are regulated by receptor activator of nuclear
factor-kappa B (RANK), its ligand (RANKL) and osteoprotegerin. The RANKL-targeted therapy has
been drawn attention as a novel bone cancer treatment in human, however little has been known
in COSA. The aim of this study was to clarify the role of RANKL and therapeutic effect of anti-RANKL
antibody on growth and tumor-induced osteolysis of COSA in a xenograft mouse model.

Materials
High-RANKL-expressing COSA cell line (CHOS; 1x105 cells/head) was transplanted into the tibia of
nude mice (n=5). Subcutaneous administration of anti-mouse RANKL monoclonal antibody was
started at the 2nd week, and mice were sacrificed at the 3rd and 5th week after transplantation.
Tissues harvested were subjected to micro-CT for bone mineral density (BMD) and
histopathological assessments.

Results
CHOS-xenografted mice showed bone osteolysis with number of OCs at the interface of the tumor
and the original bone, where strong RANKL expression was observed Furthermore, anti-RANKL
antibody administration resulted in an increase in BMD, suppression of bone osteolysis and tumor
proliferation index (PI) at the transplanted site. Notably tumor was developed in only 20% of mice
treated with antibody either at 3rd or 5th week.

Conclusions
High-RANKL expression on COSA tissues may relate to tumor-induced osteolysis through OCLs
induction, leading to more bone resorption and tumor proliferation. Thus anti-RANKL antibody
therapy may be a promising modality for COSA treatment.
 n d
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Global Gene Expression Analysis of Canine Osteosarcoma Cancer Stem Cells

Lisa Pang
(Lisa Pang, Emma L Gattenby and David J Argyle)

Roslin Institute / Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom

[email protected]

Introduction
Canine osteosarcoma is the most common primary bone tumour of dogs, accounting for 85% of
tumours originating from the bone. It is an aggressive neoplasm of older, large breed dogs and is
typically followed by lung metastasis. Osteosarcoma is also the most common primary bone
tumour of children. This is an aggressive disease with distant metastasis occurring in >80% of
individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to
contain a sub-population of cancer stem cells, which may drive tumour growth, recurrence and
metastasis. This contributes to evidence that naturally occurring canine osteosarcoma could be a
good preclinical model for the human disease.

Materials
Putative cancer stem cells (CSCs) were isolated by tumoursphere formation, and characterised by
marker expression, sensitivity to doxorubicin, and invasiveness. Isolated RNA was hybridized to the
Affymetrix Canine_2.0 gene chip. Initial data analysis was done in Partek and Ingenuity Pathway
Analysis was used for canonical pathway analysis.

Results
Here we report that CSCs can be isolated form a canine osteosarcoma cell line, KT OSA5. We show
that these cells can form tumourspheres, and are resistant to doxorubicin. Utilising the Affymetrix
canine microarray, we are able to definitively show that there are significant differences in global
gene expression profiles of isolated the cancer stem cells and the parental cells.

Conclusions
The failure of clinical therapy to eradicate osteosarcoma may be due to CSCs. Extensive analysis of
global gene expression changes will enable further elucidation of cancer stem cell biology and
identification of novel therapeutic targets.
 n d
2 WVCC 2012 - 49 –

EVALUATION OF A PANEL OF DIFFERENT ANTIBODIES TO ASSESS B CELL LINEAGE BY

IMMUNOHISTOCHEMISTRY USING A TISSUE MICROARRAY IN CANINE LYMPHOMA

Joaquim Henriques
(Joaquim Henriques, José Cabeçadas, João Matos, Fernando Constantino-Casas and Jane Dobson)

CICV- Faculty Veterinary Medicine. Lusofona University, Lisbon, Portugal

[email protected]

Introduction
Canine lymphoma it’s the most common hematopoietic neoplasm in dogs. B cell phenotype has
been associated with favorable prognostic. Commonly, CD79 epitope is used in routine
immunohistochemistry to evaluate B cell lineage

Materials
Fifty eight canine lymphomas were studied. Routine H&E and immunohistochemistry for B cell
(CD79αcγ; BLA36; PAX5; CD20) and T cell (CD3) was used to classify lymphomas according to
morphology and immunophenotype. A tissue microarray was constructed to perform
immunostaining. Normal tonsil tissue was the positive control and negative control was obtained
eliminating primary antibody. The tumor was considered positive when more than 75% off the cells
showed staining. Microscopic evaluation was made blindly 3 different times.

Results
B cell lymphomas accounted for 79% (n=46) and T cell lymphomas with 21% (n=12). B cell markers
CD79αcγ, CD20, PAX5 and BLA36 stained 69%, 74%, 65.5% and 76 % of all B cell lymphomas
respectively. A small number of tumors showed loss of at least one B cell marker. From the T cell
lymphomas 4 were CD3 + BLA36+ and two CD3+ CD20+

Conclusions
There was not a single antibody that could detect all B cell lymphomas and in some cases there was
cross reactivity between B and T cell markers. Both CD20 and PAX5 had similar performance. These
findings suggest when diagnosing and classifying B cell neoplasm based on immunohistochemistry
we should use a panel of antibodies, rather than just one (CD20 or CD79). Interestingly in our study
Pax5 did not correlate completely with CD79 as in a previous published recent study
 n d
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Evaluation of c-KIT (CD117) mutations, mRNA and protein expression in canine leukemia and
lymphoma: might c-KIT represent a therapeutical target?

Mery Giantin
(Mery Giantin, Arianna Aricò, Mauro Dacasto, Luca Aresu, Fulvio Riondato, et al)

Dept. Public Health Comparative Pathology and Veterinary Hygiene Faculty of Veterinary Medicine
University of Padua Legnaro (PD) Italy

[email protected]

Introduction
In normal hematopoietic cells, c-KIT plays an important role in proliferation, survival and
differentiation of progenitor cells. Upon maturation, its expression is lost except in mast cells,
natural killer and dendritic cells. In human hematopoietic malignancies, c-KIT is mostly expressed by
progenitor cell neoplasms and seldom by those involving mature cells. Aim of this study was to
evaluate c-KIT expression in canine hematopoietic malignancies, as there are currently no data
reported in the veterinary literature.

Materials
Sixty-nine canine lymphoid neoplasms, including 25 B-cell lymphomas (B), 21 T-cell lymphomas (T),
11 acute lymphoid leukemias (ALL) and 12 chronic lymphocytic leukemias (CLL) were enrolled. c-KIT
expression (mRNA and protein) in lymph node fine needle aspirates and peripheral blood was
measured by using quantitative Real Time RT-PCR, flow cytometry and immunocytochemistry.
Mutations on c-KIT exons 8, 9, 10, 11 and 17 were investigated by means of complementary DNA
sequencing.

Results
High amounts of c-kit mRNA were noticed in ALL, whereas CLL, B and T-lymphoma showed a
comparatively lower gene expression. Transcriptional data were confirmed at the protein level;
moreover, c-KIT localization was shown to be mostly membranous. No significant gain-of-function
mutations were observed.

Conclusions
Among canine haematological malignancies, ALL typically shows a very aggressive biological
behavior, partly being attributable to the lack of efficacious treatment options. The expression of c-
KIT in canine ALL, as evidenced by this study, may represent the rationale for using tyrosine kinase
inhibitors in future clinical trials.
 n d
2 WVCC 2012 - 51 –

MATRIX METALLOPROTEINASES AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN CANINE

LYMPHOHEMATOPOIETIC MALIGNANCIES

Arianna Aricò

Dept. Public Health, Comparative Pathology and Vet. Hygiene, Faculty of Veterinary Medicine,
University of Padua, Padua Italy

[email protected]

Introduction
Canine lymphohematopoietic malignancies are common spontaneous diseases whose biologic
behavior closely resembles their human counterparts. Matrix metalloproteinases (MMPs) and
Vascular Endothelial Growth Factor (VEGF) play a coordinated role during invasion and proliferation
of malignant cells, however little is known about their role in hematological malignancies. Aim of
this study was to investigate mRNA and protein expression of most relevant MMPs and VEGF in
canine lymphohematopoietic malignancies.

Materials
Lymph node aspirates from 26 B-cell and 21 T-cell lymphoma and peripheral blood samples from 11
cases of acute lymphoblastic leukemia (ALL) and 12 of chronic lymphocytic leukemia (CLL) were
collected. MMP-9, MMP-2 and VEGF-A protein expressions were evaluated by immuno-
cytochemistry, while MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164
mRNA levels using quantitative RT-PCR.

Results
Higher MMP-9 and VEGF-A values were noticed at mRNA and protein level in leukemia versus
lymphoma dogs and in T-cell versus B-cell lymphoma. Furthermore, MT1-MMP, TIMP-1 and RECK
mRNA amounts were significantly higher in T-cell lymphoma respect to B-cell counterpart. In the
leukemic group, TIMP-2 mRNA was significantly over-expressed in ALL. Positive and significant
correlations were found between MMP-9 and TIMP-1, and MMP-9 and VEGF-A in CLL and in T-cell
lymphoma, then among MMP-2, MT1-MMP and TIMP-2 in T-cell lymphoma.

Conclusions
The present study suggests a potential role of MMP-9, MT1-MMP, TIMP-1, TIMP-2 and VEGF in
tissue migration, angiogenesis and anti-apoptotic activity. Our results show that expression of these
markers is associated with type of leukemia (acute versus chronic) and immunophenotype of
lymphoma (B versus T).
 n d
- 52 - 2 WVCC 2012

APPLICATION OF HAN’S ALGORITHM TO CLASSIFY DIFFUSE LARGE B CELL LYMPHOMA (DLBCL) IN

DOGS

Joaquim Henriques
(Joaquim Henriques, José Cabeçadas, João Matos, Fernando Constantino-Casas and Jane Dobson)

CICV-Faculty of Veterinary Medicine, Lusófona University, Lisbon, Portugal

[email protected]

Introduction
DLBCL is the most common B cell neoplasia in dogs. Thought as a single entity, nowadays gene
expression profile identified two different prognostic important subgroups: Germinal Centre (GC)
and, Non Germinal Centre (nGCB). Imunophenotype may be used as a surrogate for this
subclassification.

Materials
Fifty eight canine lymphoma were studied. H&amp;E and immunohistochemistry for B cell and T
cell were used to classify lymphomas. A tissue microarray was constructed to perform further
immunostaining using previously validated anti-Bcl6; anti-CD10 and anti-MUM-1 antibodies. The
algorithm was applied as follows: GC tumors were CD10+ or being CD10- show BCL6+ and MUM1-.
Non GCB were all tumors that being CD10- alone or in conjunction with BCL6 are both BCL6+ and
MUM1+. Positive and negative controls were used.

Results
B cell lymphomas accounted for 79% (n=46) and from these 50% (n=23) were DLBCL.BCL6 staining
was negative in 100% of cases, although staining pattern was obtained in normal lymphoid tissue.
From the 46 DLBCL, only one was MUM1+.

Conclusions
This is the first attempt in veterinary oncology to apply this algorithm to canine DLBCL. Hans
algorithm could not be applied to our series. This may be due to: 1) lack of specificity of the
antibodies used, although this seems unlikely since we had the expected staining pattern in normal
tissue; 2) the possibility that canine DLCBL subtypes, if they exist, cannot be predicted by the
present algorithm. Further studies using gene expression profiles are needed to obtain other GC
and non GC markers for canine B cells.
 n d
2 WVCC 2012 - 53 –

The drug efflux carrier MDR1 influences efficiency and tolerability of the cytostatic treatment of
malignant lymphoma in dogs.

Irina Gramer
(Irina Gramer, J Geyer and Martin Kessler)

Institut für Pharmakologie und Toxikologie der Veterinärmedizin; Tierklinik Hofheim, Gießen,
Germany

[email protected]

Introduction
In veterinary medicine tumor therapy intends life-prolongation by conserving life quality.
Cytostatic-induced adverse effects and a developing drug resistance influence therapy success.
Drug carriers, such as the multidrug resistance carrier MDR1, play an important role for drug
resistance and tolerability. This dissertation tries to correlate cytostatic-induced side effects with
hitherto unkown MDR1 polymorphisms and correlates drug-resistance with the individual MDR1
expression levels of patients.

Materials
22 dogs with malignant lymphoma were treated in the animal hospital of Hofheim during one year.
Adverse effects were evaluated by owner-questioning and by monitoring blood values. By using
blood samples it was possible to sequence full-length PCR products which were used to identify
novel polymorphisms. Quantitative real-time RT-PCR was run to evaluate MDR1 expression during
therapeutic cycles.

Results
In this study, we identified nine novel MDR1 polymorphisms. In order to analyse their functional
relevance transport assays are planned. Furthermore, we showed that patients suffering from
severe side effects tend to have low MDR1 expression levels. In contrast, drug resistance went in
parallel with an increase of MDR1 expression. For all dogs developing drug resistance a cut-off
value could be defined for the MDR1 expression level. This value differs between dogs in tumor
progression and remission.

Conclusions
Determination of MDR1 expression levels could be a useful tool in predicting drug tolerability and a
developing drug resistance in dogs. Further studies are needed to verify this data in larger patient
groups. The novel MDR1 polymorphisms identified in this study have to be functionally
characterized.
 n d
- 54 - 2 WVCC 2012

Cytokine profiling of Bernese Mountain dogs with disseminated histiocytic sarcoma

Lise Nikolic Nielsen

(Lise Nikolic Nielsen, Mads Kjelgaard-Hansen and Annemarie T Kristensen)

Dept of Small Animal Clinical Sciences, Faculty of Life Sciences University of Copenhagen, Denmark

[email protected].

Introduction
Hypercytokinaemia has been detected in cancer in people and serum cytokines are considered
promising biomarkers of cancer staging and prognostics. Cytokine profiling has been documented in
dogs with cancer both as cytokine release and genetic expression. Cytokine concentrations in
disseminated histiocytic sarcoma (DHS) in dogs are however only sparsely investigated. The aim of
this study was to evaluate the immunological status of untreated Bernese Mountain dogs (BMD)
with DHS by assessing multiple serum cytokines and to correlate these with other inflammatory
markers.

Materials
As a prospective case control study, 17 BMD with DHS participated along with 16 healthy controls
(12 BMD and 6 dogs of various breeds). Blood samples were examined for fibrinogen, C-reactive
protein (CRP), white blood cell count, monocyte count and the following cytokines: interleukin (IL)-
6, IL-10, IL-12, IL-15, IL-18, tumour necrosis factor and monocyte chemotactic protein (MCP)-1.
Between group comparisons were done by Mann-Whitney test. Correlations tested by Spearman?s
correlation test.

Results
Significant differences for BMD with DHS were observed for ([range controls], [range BMD with
DHS]): Fibrinogen ([1.6-4.0mg/L], [2.0-12.3mg/L], P=0.002), CRP ([0-16.8mg/L], [0-379.0mg/L], P=
0.02) and MCP-1 ([107.1-1816pg/mL], [131.2-50000pg/mL], P=0.004). No significant correlations
were observed.

Conclusions
BMD with DHS have an activated inflammatory response and an increased concentration of MCP-1.
The implications of an increased MCP-1 in DHS remain to be investigated.
 n d
2 WVCC 2012 - 55 –

Clinical trial in canine osteosarcoma with the oral biphosphonate clodronate

Gerard Rutteman

Vet Spec Ctr De Wagenrenk & Utrecht University Clinic of Companion Animals, Utrecht,
Netherlands

[email protected]

Introduction
Biphosphonates can inhibit osteolysis, but also growth of cancer cells by various pathways.
Sometimes owners of dogs with osteosarcoma (OSA) deny surgical or radiotherapeutic or classical
cytostatic treatment, and other means of palliation such as a potential beneficial effect of
biphosphonates are worthwhile of investigation.

Materials
Dogs with primary OSA (19) or metastatic OSA (1) for which other treatment was considered
unwanted, were selected for this trial. Risk of pathological fractures was assessed not imminent,
and plasma creatinine values were less than 25% above upper limit. Clodronate was administered
at 10-12.5 mg/kg 2dd until significant side effects or detoriation occurred. Response was assessed
by pain relief; reduction in size; radiographic decrease in size/osteolysis.

Results
Gastrointestinal toxicity (grade 1) occurred once, and creatinine-values did not increase during
treatment. Treatment was halted within 3 weeks in 3 dogs with rapid progression, too rapid to
value antitumour effects. Six of the other 17 dogs experienced some palliation with reduction (n=1)
or absence of increase of pain (n=5) for more than 4 weeks. Response based upon tumor size was
seen in 6 dogs (SD 5, PR 1) and in 9 dogs examined radiographically in 4 dogs (SD 3, PR 1). Survival
in these 17 dogs varied from 30-380 (median 103) days.

Conclusions
While side effects were minimal, effects of clodronate upon the clinical signs of disease were
modest (SD 5 dogs, PR 1). Two dogs had longlasting pain-palliation and survival > 1 year.
 n d
- 56 - 2 WVCC 2012

Treatment of dogs with carboplatin: excretion, contamination of the environment and uptake by
owners

Tine Janssens
(Tine Janssens, Elke E Brouwers, Johan de Vos, Jan H Schellens and Jos H Beijnen)

Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer

Institute, Amsterdam, The Netherlands

[email protected]

Introduction
The use of oncolytic drugs in dogs and cats leads to a rising concern about safe handling of these
drugs and treated animals. Therefore, it is of interest to investigate the potential exposure of pet-
owners to these drugs.

Materials
Platinum was monitored as a marker in urine, faeces, saliva, sebum and cerumen of dogs. Platinum
was also measured in environmental samples and urine of the owners. Samples were collected
during 3 weeks after the first carboplatin dose (300 mg/m2). Inductively coupled plasma mass
spectrometry was used, which allows us to monitor platinum at ultratrace levels.

Results
The bulk of platinum was excreted during the first 4 days (D) after administration: urine (D1: 79.5;
D4: 2.27 mg/L), faeces (D1: 0.168; D4: 0.408 ?g/g), saliva (D1: 0.658; D4: 0.064 mg/L), sebum (D1:
65.3; D4: 37.1 pg/cm2) and cerumen (D1: 6.64; D4: 0.588 ng/sample). However, platinum was
found in all samples throughout the duration of the remaining sampling period. Environmental
samples showed a slight increase in platinum contamination in nearly all measured spots, including
those unreachable for the dog (0.24-30.1 pg/cm2). Urine samples from the owners showed normal
platinum background variation.

Conclusions
Platinum is mainly excreted during the first 4 days after administration, via all measured routes.
Environmental contamination was found, caused by the dog and by dispersion via the owners. As a
result, surfaces deemed ‘safe’, can contain platinum. Nevertheless, urine samples of the owners did
not indicate uptake of platinum. Further research is focused on investigating the relevance of these
results.
 n d
2 WVCC 2012 - 57 –

Efficiency and cytotoxicity of Nanoparticle-mediated transfection methods for DNA expression

plasmids as basis for cell therapeutic approaches

Maria Carolina Duran Graeff

(Maria Carolina Duran Graeff, Jessika-M V Müller, Karsten Feige, Saskia Willenbrock, Annette
Barchanski et al.)

PhD Student, Equine Clinic, University of Veterinary Medicine Hannover, Germany

[email protected]

Introduction
For exogenous DNA transfections reproducibly high transfection rates with low methodology-
induced cytotoxic side effects are essential to attain required effects on targeted cells. Different
approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to
increase transfection efficiencies, but data comparing toxicity of these approaches with
conventional methods are lacking.

Materials
A canine cell line was transfected with two eukaryotic expression vector constructs applying six
transfection protocols: conventional transfection reagent (FuGENE HD; FHD), FHD plus two sizes of
laser-generated AuNPs (PLAL AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP),
and two magnet-assisted transfection protocols. 24h post-transfection efficiencies were analysed
using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell
proliferation and percentage of propidium iodide (PI%) positive cells. Recombinant protein
expression was evaluated by immunofluorescence.

Results
The efficiency of pIRES-hrGFPII-eIL-12 transfections was significantly increased using AuNPs (FHD:
16%; AuNPs: 28%). PLAL AuNPs_S1/_S2 had no significant cytotoxic effect, while the Plano-AuNPs
induced higher PI% and lower cell proliferation (p<0.05). Transfection of pIRES-hrGFPII-rHMGB1
using PLAL-AuNPs resulted in high efficiency (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2:
50%, p<0.05), while magnet-assisted transfection showed lower efficiencies than FHD transfection.
The PLAL-AuNPs_S1/_S2 increased the PI% (p<0.05), but no consistent effect on cell proliferation
was observed. The magnet-assisted protocols were less effective, but showed lowest cytotoxic
effects.

Conclusions
Addition of AuNPs significantly improved the transfection efficiency, but some combinations
increased cytotoxicity depending on the type of AuNPs and used vector. For routine use of PLAL-
AuNPs, the specific nanoparticle formulation and expression plasmid combination has to be
considered.
 n d
- 58 - 2 WVCC 2012

The novel chemoprotectant drug, TavoceptTM, facilitates a shortened cisplatin infusion protocol
for treatment of cancer-bearing dogs

Carolyn J. Henry
(Carolyn J Henry, Kim A Selting, Sandra Axiak, Jeffrey N Bryan, Deborah J Tate and Hausheer Fred)

University of Missouri, Columbia, USA

[email protected]

Introduction
In order to prevent nephrotoxicity, cisplatin is currently administered to dogs over 20 minutes,
along with 6 hours of saline diuresis, making its use inconvenient in clinical practice. TavoceptTM is
an investigational new drug shown to prevent some of the common and serious toxicities
associated with platinum and taxane chemotherapy in people. Tavocept co-administration with
chemotherapy enhances antitumor activity of cisplatin in cell culture, animal studies and human
clinical trials. This study was designed to determine if TavoceptTM can facilitate the safe use of a
shortened cisplatin infusion protocol in cancer-bearing dogs.

Materials
Seven client-owned dogs with bladder cancer were treated with TavoceptTM in conjunction with
piroxicam and cisplatin. Dogs underwent a 90-minute infusion protocol utilizing TavoceptTM (18.4
g/m2 IV), cisplatin (60 mg/m2 IV), and saline (18 ml/kg/hour), repeated q21d for 4 planned doses.
Maropitant (2 mg/kg one hour before treatment) was added to the protocol after emesis was
noted with the first two enrollees. Piroxicam (0.3 mg/kg/day PO) was initiated on Day 1.

Results
The 90-minute infusion protocol was well-tolerated with the addition of maropitant. Elevation in
BUN (median = 67 mg/dl; range = 50 – 83 mg/dl) without comparable increase in creatinine or
development of isosthenuria was a consistent post-treatment finding that was generally subclinical
and improved with discontinuation of piroxicam.

Conclusions
The protocol consistently resulted in pre-renal azotemia thought to be related to piroxicam
therapy. TavoceptTM facilitates a shortened (90-min) cisplatin/saline infusion time that should be
evaluated without concurrent NSAID therapy in order to develop a more convenient clinical
protocol.
 n d
2 WVCC 2012 - 59 –

Assessment of cardiac arrythmia in dogs with cancer receiving doxorubicin : a prospective Holter
study of 20 dogs.

François Serres
(François Serres, Dominique Tierny, Laurent Marescaux, Antoine Hidalgo and Cécilia Robat)

ONCOVET Villeneuve d’Ascq France

[email protected]

Introduction
Doxorubicin is one of the most commonly used chemotherapy drugs in dogs with cancer. Its use is
limited by cumulative cardiotoxicity, which could manifest as systolic dysfunction or rhythm
disturbance. Few studies focused on the cardiac rhythm disturbance induced by doxorubicin
administration, and all relies on short duration electrocardiographic studies. Recently, a 24-hour
standardized hospitalization has been legally required for all dogs receiving chemotherapy in
France, allowing the realization of systematic Holter study in dogs receiving doxorubicin.

Materials
Dogs presented at the Oncovet center in which at least two Doxorubicin administrations was
planned were prospectively recruited for assessment of rhythm disturbance.

Results
Seventy-five Holter monitoring of 24 hours duration were performed in 20 dogs: 11 presented with
lymphoma, 6 with mammary carcinoma, 3 with sarcoma. No significant difference in mean heart
rate was observed between consecutive administrations of Doxorubicin. In 6 dogs, more than 50
ventricular premature complexes (VPC) /24 hours were observed, during the first, second, fourth
and sixth administration (in 2,1, 1, and 2 dogs respectively). At least one episode of ventricular
tachycardia was observed in three dogs. Echocardiographic examination was unremarkable in all
dogs presenting with arrhythmia.

Conclusions
A relatively high frequency of VPC was observed in 30% of dogs with spontaneous tumor receiving
doxorubicin. Rhythm disturbances were not related to cumulative doses of doxorubicin, and did not
occur concurrently with doxorubicin administration. Although severe arrhythmias were only
observed in a small subset of dogs, correlation between rhythm disturbance and survival must be
addressed in further studies.
 n d
- 60 - 2 WVCC 2012

DNA vaccination against human CSPG4 induces cross-reacting antibody response in dogs with
oral malignant melanoma expressing CSPG4

Federica Cavallo
(Federica Cavallo, Simone Prestigio, Federica Riccardo, Lorella Maniscalco, Saray Lora Mayayo et al)

University of Turin, Dept of Clinical and Biological Sciences, Molecular Biotechnology Center, Italy

[email protected]

Introduction
Melanoma is one of the most immunogenic tumors and numerous trials of immunotherapy for
metastatic melanoma have been performed, though response remains infrequent. Canine
malignant melanoma (MM) shares many characteristics with its human counterpart, making dogs a
valuable clinical model to assess efficacy of novel therapeutic strategies. Chondroitin sulfate
proteoglycan-4 (CSPG4) is a membrane-bound chondroitin sulfate proteoglycan highly expressed by
human melanoma cells and with a restricted distribution in normal tissue. We evaluated the
immunohistochemical expression of CSPG4 in canine MM and the capability of DNA electroporation
of a plasmid coding for the human CSPG4 (hCSPG4) in inducing an effective immune response in
dogs with surgically resected CSPG4-positive MM.

Materials
Dogs with biopsy confirmed CSPG4-positive oral MM and absence of detectable metastases beyond
the first regional lymph node had an en-bloc tumor excision and were then electroporated monthly
with 500 µg of hCSPG4 plasmid. Specific antibody (Ab) response was evaluated by
immunofluorescence and ELISA by using cells expressing human or canine CSPG4. T-cell response
against pools of 15 amino acid peptides covering the entire sequence of dog CSPG4 are under
investigation by ELISPOT.

Results
DNA electroporation with hCSPG4 plasmid did not cause any important local or systemic side
effects. All vaccinated dogs developed Abs against hCSPG4 cross-reacting with the canine
orthologue.

Conclusions
DNA electroporation with hCSPG4 plasmid is safe and effective to break tolerance against dog
CSPG4 and thus is potentially efficacious against canine MMs. The preliminary results regarding
survival of the vaccinated dogs are encouraging.
 n d
2 WVCC 2012 - 61 –

A NEW CONCEPT WITHIN LOCALISED CANCER TREATMENT

Thora Johanna Jonasdottir

(Thora Johanna Jonasdottir, Anders Høgset, Kristin Eivindvik, Karin Nord and Per Walday)

Norwegian School of Veterinary Science, Dept of Companion Animal Clinical Sciences, Small Animal
Section Oslo / PCI Biotech AS~ Strandveien 55~ N-1366 Lysaker, Norway

[email protected]

Introduction
Achieving intentional effect of chemotherapeutic drugs with intracellular targets depends on
sufficient delivery of the drug to the cytosol or nuclei of malignant cells. However, cytosolic delivery
is often hindered by endosomal capture of the drug. PhotoChemical Internalisation (PCI) is a
technology for light-directed drug delivery by triggered endosomal release, used to enhance the
drug effect at the cellular level in the tumour. Combining the photosensitizer Amphinex® with the
established cytotoxic agent bleomycin, a clinical phase-I/II study has been completed in humans
with promising results. The objective of the presented study was to investigate the safety and
tolerance, and to estimate the most appropriate Amphinex® dose, light administration and light
dose for PCI treatment in dogs affected with cancer.

Materials
Dogs with any external inoperable tumour of a minimum 2x2 cm could be included. Amphinex® at
dose levels of 0.25 and 0.5 mg/kg was given three to four days before administration of bleomycin,
followed by tumour light administration 2-3 hours later.

Results
Administration of Amphinex® was well tolerated. Both interstitial and superficial delivery of light
was used. Using optical fibers with cylindrical diffusors was found to be more practical than bare-
end fibers for interstitial light administration. Tumour light absorption and permeability was
variable between different tumours. Some significant tumour responses were observed.

Conclusions
In the current study, PCI treatment using Amphinex® and bleomycin was found to be well tolerated
by the dogs at the human therapeutic dose levels. When using interstitial light administration it
might be advantageous to adapt the light dose individually.
 n d
- 62 - 2 WVCC 2012

FAMILIAL AND BREED RELATED ABSTRACTS

 n d
2 WVCC 2012 - 63 –

Whole Genome Mapping of Anal Sac Gland Carcinoma Susceptibility Loci in the English Cocker
Spaniel

Jesus Aguirre-Hernandez
(Jesus Aguirre-Hernandez, Gerry Polton and David R Sargan)

Dep of Veterinary Medicine, University of Cambridge, United Kingdom

[email protected]

Introduction
Anal sac gland carcinoma (ASGC) is a tumour with an elevated incidence in English cocker spaniel
(ECS) dogs in the UK (mean OR = 7.3) and in the US (OR = 8.7), as well as in other spaniel breeds. It
has no gender bias although neutered males show slightly higher predisposition. The average age of
onset in ECS is 9.1 years ± 1.9.

Materials
Sixty three cases (diagnosed by pathology and cytology) and 141 control individuals were
genotyped using a 172 K SNP array. A mixed model association test was used to correct for cryptic
and familial relatedness, as well as for inbreeding effects. The p value required for genome wide
significance was established at 1.74E-06 by applying a Bonferroni correction for the effective
number of tests performed.

Results
A locus on chromosome 2 attained genome wide significance (P value: 7.54E-07 after accounting
for inflation, Pcorr = 0.022) and four additional loci had p values just above this significance level (P
values < 1.0E-5). These were located on chromosomes 2, 7, 28 and 32. The significant locus is within
a very small region showing linkage disequilibrium that contains a single gene. No mutations have
been found in the coding sequence of this gene.

Conclusions
A putative locus underlying the relatively high risk to ASGC in the English cocker spaniels has been
mapped, with four additional loci showing small, but not yet significant, p values. The identification
of more loci contributing to the presentation of this tumour will require genotyping additional
samples.
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Heritable Traits Influence Cell-Intrinsic Behavior of Sporadic Canine Cancers

Jaime Modiano
(Jaime Modiano, Aaron L Sarver, Milcah C Scott, Aric M Frantz, Jong Hyuk Kim, Daisuke Ito, Ashley J
Graef, Tae Hyun Hwang, Erin B Dickerson, Leslie C Sharkey, Timothy D O’Brien, Matthew Breen,
Kerstin Lindblad-Toh and William C Kisseberth)

University of Minnesota, Minneapolis, United States

[email protected]

Introduction
Breed predilections in canine cancer have been established for more than 40 years, but the
molecular basis for these associations is largely unknown. Previous work from our groups showed
that recurrent aberrations in tumor genomes are peculiarly associated with specific breeds. We
thus hypothesized that breed-specific (heritable) traits would influence not only the genetic
complement, but also the biological behavior of canine tumors.

Materials
We used genome-wide gene expression profiling of tumor tissues and isolated tumor cells
(expanded in culture) to define molecularly distinct groups of canine lymphoma, osteosarcoma, and
hemangiosarcoma. Differences were validated using quantitative real-time RT-PCR; correlations
between steady state levels of mRNA and protein were established using immunoblotting,
immunohistochemistry, and functional assays.

Results
Unsupervised analyses of cultured cells derived from hemangiosarcoma and osteosarcoma showed
robust grouping according to breed. Enriched genes were detectable as functional proteins, which
in turned altered cellular behavior in vitro. However, the breed-associated signatures were masked
in whole tissues; xenograft experiments suggest this is due to extensive tumor infiltration by
stromal cells, diluting the intrinsic profiles of the isolated tumor cells.

Conclusions
The data indicate heritable traits modulate gene expression and biological behavior of tumor cells.
However, these breed-specific traits are likely retained as “genetic relics” in advanced tumors,
where the influence of the tumor microenvironment may be a more powerful determinant of gene
expression and biological behavior. We propose that systematic integration of multiple genome-
wide datasets will reveal the significance of heritable traits in early events associated with cellular
transformation and progression of sporadic tumors.
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2 WVCC 2012 - 65 –

Identification of predisposing genetic risk factors in canine cancer

Malin Melin
(Malin Melin, Arendt Maja, Ivansson Emma, Elvers Ingegerd, Karlsson Elinor, Tonomura Noriko,
Sigurdsson Snaevar and Lindblad-Toh Kerstin)

Science for Life Laboratory, Uppsala University Uppsala Sweden

[email protected]

Introduction
Cancer is one of the most prevalent diseases in both humans and dogs and specific breeds often
show a predilection for certain tumour types. The molecular basis of the increased cancer risk in
the breeds is mostly unexplained and knowledge about the susceptibility genes may enable
improved diagnosis and treatment. We have focused on identifying predisposing genetic risk
factors for a selection of tumour types, including mammary tumours, osteosarcoma, lymphoma and
mast cell tumours. A few high-risk breeds have been chosen for initial investigations of each
tumour type.

Materials
We have collected large case-control materials with in-depth clinical information about each dog.
The sample collection has been performed both in Europe (as a part of the LUPA consortium) and in
the US by extensive collaborations. Using these cohorts, we have performed genome-wide
association studies (GWAS) in a few hundred dogs per tumour type using 170,000 SNPs.

Results
For each tumour type we identify multiple significant loci associated with tumour development. We
mostly find that the associated loci differ between tumour type, breeds and sometimes even
between the US and European populations of a single breed. Targeted resequencing has revealed a
large number of candidate mutations that are currently being validated and assessed for
functionality.

Conclusions
We have identified a large number of candidate genes for canine cancer. The genes are now being
investigated further in dogs and human cancer patients. By utilizing the comparative aspects, this
could provide a unique opportunity to improve diagnosis and treatment of cancer in both dogs and
humans.
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Familial nonmedullary thyroid carcinoma in the German Pointer longhair.

Johan de Vos
(Johan de Vos and Liesbeth van der Waaij)

De Ottenhorst, Veterinary Oncology Referral Centre, Animal Hospital Zeeuws-Vlaanderen

Terneuzen / Animal Breeding and Genomics Centre, Wageningen University, Netherlands

[email protected]

Introduction
In humans, familial medullary and non-medullary thyroid cancer (FMTC/FNMTC) accounts for <10%
of thyroid tumours. FMTC is mainly caused by dominantly inherited RET-mutations. The genetic
basis of FNMTC, primarily papillary and follicular carcinoma, is unknown. Canine FMTC is described
in Alaskan Malamutes. Our study describes FNMTC in the German Pointer longhair (GPL).

Materials
A clinical-pathological and pedigree analysis was performed on 23 GPL with thyroid tumours.
Diagnosis was made through cytology or histology.

Results
Eleven dogs had unilateral, twelve had (a)synchronously developing bilateral tumours with
metastases in two dogs. Median age of diagnosis was 7.5 years (range 4.5-13). The cystic tumours
gradually progressed over 0.5-2 years. Sixteen dogs underwent surgery. Uniform histopathological
diagnosis was follicular carcinoma. Five dogs died due to loco-regional disease, five deaths were
non-tumour related. The affected dogs had 7 common ancestors. In a litter of four, two females
had thyroid cancer. One had 5 litters herself. The first three with the same male, the other two had
a father that was also, respectively, full brother and father of two other affected dogs. The ratio
“affected to total born” in litters #1-3 was 0.33 (4/12), 0.11 (1/9), and 0.46 (5/11). In litter #4 the
ratio was 0.29 (2/7), and in litter #5, four years of age at time of analysis, 0.20 (1/5).

Conclusions
At least a subgroup of follicular thyroid carcinomas in the GPL is heritable. The suggested mode of
inheritance is monogenic autosomal recessive. A follow-up study to determine the genetic cause
will be performed.
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POSTERS
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The UK experience with the melanoma vaccine: use of OnceptTM in 32 dogs with oral melanoma

Elisabetta Treggiari
(Elisabetta Treggiari and Susan M North)

VRCC, Essex, UK

[email protected]

Introduction
A human tyrosinase DNA vaccine, ONCEPT™, has been shown in previous studies to be safe and
effective against melanoma by inducing both a T-cell and humoral response against malignant
melanocytes. We report here on the first European experience of the vaccine.

Materials
Medical records were examined for 32 dogs seen at VRCC between 2009-2011, 8 are still under
treatment. Nineteen male and thirteen female dogs, with a mean age of 10.4 years, were referred
for treatment of oral malignant melanoma with the vaccine. Local control prior to vaccination was
achieved with either surgery and/or radiotherapy. In some cases surgery had been carried out by
the local veterinary surgeon prior to referral for the vaccine.

Results
Overall MST was 270 days, with a median survival time of 180 days for stage I (range 163-700), 402
for stage II (range 60-485), 270 for stage II-III (range 87-645), 189 for stage III (range 60-428). Limits
of the retrospective study induced us to classify some patients in stage II-III, when no initial
measurement of the mass was available.

Conclusions
Use of the vaccine appears to be safe and successful in the management of oral melanoma. Eight
dogs are still under treatment and received a full vaccination course, including those who already
had their first booster. Stage I and stage II patients are still alive over 2 years (range 455-820 and
270-880 days, respectively).
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2 WVCC 2012 - 69 –

Primary cell culture and characterisation of a canine hepatocellular carcinoma

Adam George Gow

(Adam George Gow, Lisa Yan-Toi Pang, Linda Morrison and David J Argyle)

The Roslin Institute / Royal (Dick) School of Veterinary Studies, Edinburgh, UK

[email protected]

Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver tumour in dogs. Although
surgery may be curative, as HCC may be diffuse, multifocal and have metastasis, this is not always
an option. Chemotherapy has not yet been shown to improve survival in canine HCC. We aimed to
develop an in-vitro cell culture model to aid study of Canine HCC further, and investigate the
existence of a stem-cell fraction, which may be a useful target for therapy.

Materials
A 1cm3 section of a tumour was collected post-mortem from a 15 year old cross-breed dog. After
digestion with collagenase, fragments were cultured in modified Eagle’s medium with 10% FCS, L-
glutamine and antibiotics. The resultant adherent cells were characterised by PAS staining and
immonocytochemistry for glycogen, and keratin 7 (C7) respectively. The stem cell characteristic of
ability to form spheres in anchorage-independent, semi-solid conditions was also investigated.
CD133 , a cancer stem cell marker, was used for magnetic assisted cell sorting (MACS).

Results
A pleomorphic cell population was isolated. None were positive for glycogen, suggesting this was a
poorly differentiated HCC. C7 staining was negative, consistent with previous reports. Adherent
cells showed the ability to form spheres and could be passaged up to 3 times. Based on MACS, 1.5%
of the adherent cell population was CD133 positive.

Conclusions
A putative stem cell fraction has been identified from the primary cell culture. Further work will
characterise the CD133+/- cells, compare their ability to form spheres, chemosensitivity and also
metastatic potential by invasion assay.
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SERUM VITAMIN D LEVELS IN CANINE MAMMARY TUMOR PATIENTS AND HEALTHY DOGS

Simone Crestoni Fernandes

(Simone Crestoni Fernandes, Paulo Roberto Del Valle, Maria Aparecida, Azevedo Koike Folgueira,
Lúcia Maria, Guedes Silveira, Keiji Sato and José Guilherme)

College of Medicine - Universidade de São Paulo (USP), Brazil

[email protected]

Introduction
Vitamin D in dogs is mainly obtained from food sources, since there is little cutaneous synthesis,
however no comprehensive data concerning 25(OH)D3 serum levels in healthy dogs is available.
There is evidence that higher serum 25(OH)D3 or 1,25(OH)2D3 levels may reduce the risk of many
types of cancer in human beings, including breast cancer. Vitamin D may be involved in the control
of proliferation, differentiation and apoptosis of mammary cells. Thus, our goal was to compare
serum 25(OH)D3 levels in healthy dogs and dogs with mammary tumors in the region of São Paulo
city, Brazil.

Materials
Serum samples were collected from 28 female dogs with mammary carcinoma and 64 dogs without
tumor (50 females and 14 males, control group). 25(OH)D3 serum concentration was determined
using a competitive radioimmunoassay (DiaSorin S.A, Stillwater/MN, USA).

Results
Approximately 37% of female (both tumor-bearing and healthy) and 21% of male dogs presented
serum 25(OH)D3 insufficiency (&lt;19 ng/mL). No difference was detected in 25(OH)D3 serum levels
from carcinoma bearing animals (27,67 +/- 13,98 ng/mL) as compared with dogs from the control
group (24,95 +/- 12,72 ng/mL) (Student t test). An indirect correlation between 25(OH)D3 serum
concentration and animal age was detected in tumor bearing (benign or malign) patients (n=39; r= -
0,327; Pearson correlation; p=0,042).

Conclusions
In conclusion, a relative 25(OH)D3 insufficiency was detected in a considerable fraction of healthy
and sick dogs, however no difference in 25(OH)D3 serum level was detected between the groups.
Our results suggest that vitamin D supply should be re-evaluated in commercial pet food.
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2 WVCC 2012 - 71 –

Oral neoplasms in dogs: a 15-year retrospective study

João Requicha
(João Requicha, Maria dos Anjos Pires, Carlos Manuel Albuquerque and Carlos Alberto Viegas)

University of Trás-os-Montes e Alto Douro, Portugal

[email protected]

Introduction
Oral proliferative lesions are common in domestic carnivores. The oral cavity is the fifth most
common site for neoplasia in the dog. The exact nature of any neoplasia can only be determined by
histopathological examination.

Materials
At the University of Trás-os-Montes e Alto Douro a retrospective study of the canine oral cavity
tumours was performed from January 1995 until December 2010, considering biological behaviour,
gender, age and breed as variables.

Results
From 180 tumours, 116 benign (64.4%) and 64 malignant neoplasias (35.6%) were reported. Among
the benigns, 107 (92.2%) were epulides (63 fibromatous epulides, 32 ossifying epulides, 11
acanthomatous epulides and 1 giant cell epulis), 6 papillomas (5.1%), 2 fibromas (1.7%) and 1
ameloblastoma. Within the malignant, 50% were melanomas (25 pigmented and 7 amelanotic),
31.3% fibrosarcomas, 10.9% squamous cell carcinomas and 4.7% lymphomas, and 1 mast cell
tumour. After statistic evaluation and discarding any analyses without data, it was observed that
neoplastic lesions had a higher incidence in males (58.9%) than in females (36.7%), and in 10-year-
old animals (22 cases). Benign tumours had higher reporting between 2 and 8 years of age, whereas
malignant tumours occurred mostly in dogs over 8 years of age. Regarding the breed, the most
affected were mongrel dogs (28.9%), followed by the Boxer (26.7%) and the Poodle (5.6%), among
26 different breeds.

Conclusions
This work provided additional information to describe this type of disease, different from that
obtained in other published studies.
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Radiation therapy in dogs with intracranial neoplasms

Irene Flickinger
(Irene Flickinger, Birgitt Wolfesberger, Miriam Kleiter, Akos Pakozdy and Michael Leschnik)

Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine, Oncology
Service, Vienna, Austria

[email protected]

Introduction
Radiotherapy is the treatment of choice for inoperable intracranial tumors. The purpose of this
retrospective study was to evaluate the efficacy of radiation therapy in dogs with intracranial
neoplasms.

Materials
Dogs were examined for the following inclusion criteria: (1) focal brain lesions identified by
advanced diagnostic imaging (CT, MRI) consistent with neoplastic disease, (2) daily radiotherapy
with 6 MV to a total dose of 45 or 48 Gray. Medical records were reviewed from 2006-2010 to
identify signalment, clinical signs, tumor site and type, survival time and cause of death.

Results
Thirty eight dogs with a median age of 8 years (range 2-13) with a mean weight of 22.5 kg were
analyzed. Generalized seizures were the most common presenting sign (45%). Head tilt (11%),
ataxia (18%) and vision deficits (16%) were also observed as was PU/PD (24%) in endocrine active
pituitary tumors. Seventeen lesions were classified as extraaxial, fifteen as hypophyseal and six
were intraaxial. Corticosteroids (90%) and antiepileptics (50%) were frequently used for
symptomatic treatment. Median overall survival time was 579 days and the 1-, 2- and 3-year
survival rates were 63%, 34% and 18%, respectively. The cause of euthanasia was most likely
tumor-related in 21 dogs.

Conclusions
In this study radiotherapy achieved a median overall survival time of 579 days in dogs with the
diagnosis of a primary intracranial tumor based on advanced imaging. As CT- guided biopsies and
neurosurgery become more routinely offered, future studies might identify further prognostic
factors and improve survival times by the use of combination therapies.
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2 WVCC 2012 - 73 –

PHENOTYPE REFERENCE DATA OF LYMPH NODE ASPIRATES FROM HEALTHY DOGS

Barbara C. Rütgen
(Barbara C. Rütgen, Ramona König, Ilse Schwendenwein, Sabine Essler, Sandra Groiss and Armin
Saalmüller

Central Laboratory, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna,

Austria

[email protected]

Introduction
Canine lymphoma is the most common spontaneously occurring haematopoietic neoplasia in dogs.
Recently, immunophenotypic methods have been established for diagnosis and classification. In
this study popliteal lymph nodes from 25 dogs euthanized due to various diseases except
hematopoietic malignancies were analysed in order to establish laboratory reference data for
lymphocyte populations in lymph nodes.

Materials
Lymph nodes were homogenised into single cell suspensions. Cytospin preparations were stained
with DiffQuick® and examined microscopically for lymphocyte morphology. The expression of
leukocyte differentiation antigens was determined with canine-specific monoclonal antibodies and
analyzed by flow cytometry. Additionally, polymerase chain reaction for antigen receptor
rearrangement of B- and T-cell clonality was performed.

Results
Cytology revealed a mixed lymphocytic population in all samples confirming the tumour free status
of the material. Lymphocyte populations showed following percentages and ranges of positive cells:
CD3 (52.8±24.1%), CD3-12 (57.3±14%), CD4 (32.2±9.4%), CD5 (53.5±12.9%), CD8 (14.8±7.2%),
CD11a (87.5±16.8%), CD14 (6.5±6.5%), CD21 (30.8±11%), CD56 (5.2±6.5%), CD79αcy (46.5±13.6%),
TCR-γδ (3.8±2.9%). All samples exhibited a non-clonal pattern for TCRγ and IgH by showing faint
ladders or smears indicating a non-tumourigenic status for the respective samples.

Conclusions
Results for lymphocyte subsets from normal canine lymph nodes are in accordance with former
published data. Additionally, we have established the first reference data for CD3-12, CD5, CD11a,
CD56 and CD79αcy. Our data facilitate differentiation between benign and malignant lymphocyte
populations and will help to get further insight into different reactive patterns.
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Expression of mammalian target of rapamycin (mTOR) in feline mammary tumors: a potential

target therapy in triple negative cancer

Lorella Maniscalco
(Lorella Maniscalco, Selina Iussich, Mauro Denina, Bartolomeo Biolatti, Raffaella De Maria et al)

Dep of Animal Pathology, University of Turin

[email protected]

Introduction
Triple-negative breast cancer (TNBC) is defined by the absence of the ER, PR and HER2 receptors.
mTOR is a highly conserved serine/threonine kinase activated in several types of cancer and it is the
target for therapy with rapamycin. mTOR is significantly over-expressed in TNBC and may be a new
target for the treatment of these types of tumours instead of hormonal therapy and HER2
inhibition. Feline mammary carcinomas (FMC) are frequently negative to these hormonal receptors
and thus they represent an excellent model for the non-hormone dependent breast cancer. Aim of
this work is to evaluate the expression of mTOR in FMC samples and in primary feline mammary
cell lines in comparison with the ER/PR/HER2 phenotype

Materials
Expression of mTOR, ERα, PR and HER2 was evaluated by immunohistochemistry on 11 benign
lesions, 27 FMC, 4 metastasis and 2 normal mammary gland and by Western blot on 6 FMC cell
lines. Queens were followed up over 24 months after surgical treatment.

Results
mTOR positivity was found in the 27% (3/11) of benign lesions, in the 51% (14/27) of FMC and in all
metastasis analyzed. Western blot analysis revealed that 4/6 expressed mTOR. All triple negative
FMC (TNFMC) analyzed (9/27) showed a strong mTOR immunolabelling. No correlations with
clinical follow up was found.

Conclusions
mTOR plays an important role in the pathogenesis of FMC and its high expression in TNFMC
confirms FMC as suitable model for the development of new therapeutic strategies in comparative
oncology
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2 WVCC 2012 - 75 –

THERMOGRAPHIC CHARACTERIZATION OF CANINE MAST CELL TUMOR (MCT) – Preliminary Study.

Samanta Rios Melo

(Samanta Rios Melo, Thaís Rodrigues Macedo, Marcos Ishimoto Della Nina and Julia Maria Matera,)

Department of Surgery – School of Veterinary Medicine and Animal Science – University of São
Paulo, Brazil

[email protected]

Introduction
MCT have a variable appearance, prognosis and surgical assessment. Thermography shows a non-
invasive diagnostic method, based on relationship between inflammation, blood flow, and disease.
Thermography has been used in medicine in areas such as orthopedics, cardiology, rheumatology
and oncology. The FDA approved thermography as a diagnostic method to breast cancer patients.
The focus of this study was to characterize the MCT based on thermographic images.

Materials
Were analyzed 12 thermographic images of canine MCT. Thermal camera FLIR b400 was used and
Flir Quick Report Software to evaluate the temperature images: -total image, central area and three
area margins from the central point of the tumor (1, 2 and 3 cm).

Results
The mean temperature of all tumors, in their middle point and central area was 31.73°C and
31.86°C, respectively. The non-ulcerated tumors had shown a median temperature in their middle
point and central area of 31.39°C and 31.31°C. The ulcerated tumors had 32.43°C and 32.95°C,
respectively. For all them the Mean temperature of Marginal Area 1 (MMA1) was 31.85°C; the
MMA2 was 31.86°C and the MMA3 was 32.04°C. From the 12 tumors, 50% were classified as
histological grade II and 50% is unknown at this point of the study.

Conclusions
The ulcerated tumors had shown a smallest central temperature than the ulcerated ones, and the
temperature of the tumors margins tends to increase as far as we go from the center of the tumor.
These preliminary findings demonstrate a new possible tool for surgical planning, characterizations
of invasiveness and activity of the MCT.
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Firocoxib palliative treatment in dogs with end stage cancer: Impact on their Quality of Life

Hugo Gregório
(Hugo Gregório, Justina Prada, Isabel Pires and FelisbinaLuísa Queiroga)

Hospital Veterinário Porto, Portugal

[email protected]

Introduction
Assessment of Quality of Life (QoL) has gained much attention in the human cancer field and more
recently in the veterinary field where the first questionnaire for its evaluation has just been
presented (Lynch et al. 2011). Although survival outcomes are still the major criteria when defining
treatment plans for veterinary cancer patients , the fact that animal owners are probably much
more sensitive to QoL issues raises its importance, especially in the palliative setting. Present work
aims to evaluate the impact of Firocoxib (a specific Cox-2 inhibitor) in the QoL of dogs with end
stage cancer.

Materials
Owners of 13 animals with different end stage cancers were asked to answer a 24 questions
questionnaire regarding different aspects of QoL at day 0 and 14 days after treatment with
firocoxib 5 mg/kG PO SID as the only treatment.

Results
Significant improvements (p<0.05) were recorded in 13 of the 23 questions regarding several
aspects of QoL: happiness, mental status, pain, appetite and mobility. No significant differences
were recorded in hygiene habits and water consumption. Overall QoL assessed by the owners was
also significantly improved (p<0.01).

Conclusions
The use of firocoxib significantly increased overall and several particular dimensions of QoL in
animals with different end stage cancers. These results will encourage to evaluate the effects of
firocoxib on long term use and in specific tumour types in the future either as a single palliative
treatment agent, or in combination with other cytostatic agents.
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2 WVCC 2012 - 77 –

KIT receptor tyrosine kinase dysregulation in feline cutaneous mast cell tumours

Silvia Sabattini
(Silvia Sabattini, Marta Guadagni Frizzon, Fabio Gentilini, Giuliano Bettini and Maria Elena Turba)

University of Bologna - Department of Veterinary Medical Sciences, Italy

[email protected]

Introduction
Feline cutaneous mast cell tumours (cMCTs) are characterized by a variable biologic behaviour.
Development of multiple cutaneous nodules and concurrent visceral involvement, along with
inconsistency of conventional prognostic aids, justify present uncertainty in differentiating benign
from malignant forms. c-kit proto-oncogene activating mutations have been reported in 68% of
feline MCTs, however their prognostic relevance has not been determined. This is a prospective
study performed on feline cMCTs with variable clinical presentation to assess whether KIT
immunohistochemical overexpression can be regarded as indicative of c-kit mutations, and
evaluate their relationship with disease progression.

Materials
Cats with a histological diagnosis of cMCT in the absence of visceral involvement were enrolled.
Patients underwent surgery and were followed for at least 6 months. KIT/CD117
immunohistochemistry and c-kit mutation analysis (exons 8,9,11) were performed on tumour
samples and correlated with DFI and OS.

Results
Twenty-one cats with 11 solitary, 4 multiple, and 6 disseminated cMCTs were included.
Determinations were performed on 28 tumour samples. KIT cytoplasmic expression was observed
in 20 tumours, showing a shorter DFI (P=0.013) and OS (P=0.04). c-kit encoding mutations were
detected in 58% of cases (exon 8, 12%; exon 9, 46%; exon 11, 4%), but they were not directly
correlated with clinical behaviour.

Conclusions
c-kit mutations occur frequently in feline cMCT, and their incidence is higher among tumours with a
CD117 overexpression, but their prognostic relevance needs to be assessed in a larger series.
Multiple nodules from any one cat did not harbour the same mutations, suggesting that they may
represent separate events.
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CHEMOTHERAPY AND TELOMERASE VACCINE AS NOVEL APPROACH TO MANAGE CANINE

LYMPHOMA

Fabio Valentini
(Fabio Valentini, Alessandra Gavazza, George Lubas, Tommaso Furlanello, and Gennaro Ciliberto,)

Dip. Clinica Veterinaria, Università di Pisa, Italy

[email protected]

Introduction
Canine malignant lymphoma (ML) is managed with different chemotherapy regimens. The ability of
Adenovirus (Ad6) and DNA electroporation (DNA-EP) based vaccine to induce immune responses
against dog Telomerase (dTERT) and its impact on survival has been evaluated.

Materials
Among a population of dogs affected by ML, twenty animals with high-grade ML, B-cell-type,
generalized lymphoadenopathy, stage III-IV, were enrolled (Chem/Vax). Dogs were treated with
cyclophosphamide, vincristine and prednisone for 6-8 weeks to reach clinical remission (CR). Then
maintenance chemotherapy (MC) was instituted (prednisone and/or chlorambucil and melphalan
plus vincristine) according to our internal protocol. Nineteen dogs served as controls using
chemotherapy only. Some dogs of both groups relapse ML and doxorubicin was used to re-induce
CR and then returned to MC. Chem/Vax dogs during MC in CR received two Ad6-dTERT injections
and then treated with 5mg of dTERT-DNA followed by DNA-EP (three times). Additional series of
DNA-EP were performed months later according to immunological and clinical conditions. The
dTERT-specific immune response was tested and clinical and blood parameters have been
recorded. The median overall survival time (MOST) was evaluated.

Results
Almost all animals developed dTERT-specific immune response, which remained detectable with
absence of apparent autoimmunity or other side-effects. The MOST of Chem/Vax dogs was
significantly increased over controls (82 vs 37 weeks, respectively, p=0.000141).

Conclusions
Ad6/DNA-EP-based cancer vaccine against dTERT induces long-lasting host immune response, can
be combined with standard chemotherapy, and significantly prolongs the survival of ML canine
patients.

*Study approved by Ministry of Health, Italy

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2 WVCC 2012 - 79 –

Value of the RECIST method to evaluate clinical response to neoadyuvant treatments in canine
mammary tumours

Víctor Domingo
(Víctor Domingo, Eva Rollón, Javier Andrés, Silvia Guil-Luna, Raquel Sánchez-Céspedes, Yollanda
Millán and Juana Martín de las Mulas)

Clinica Veterinaria Recuerda, Granada, Spain

[email protected]

Introduction
RECIST (Response Evaluation Criteria in Solid Tumours) (Therasse et al., 2000) is the method most
widely used in human oncology to evaluate the clinical response (ClinR) to pharmacological
treatment. The purpose of this work was to analyze the value of RECIST in bitches with mammary
carcinoma treated with the progesterone receptor (PR) antagonist aglepristone.

Materials
Fifty six entire bitches with mammary carcinoma were treated with 40 mg/Kg aglepristone (47) or
placebo (9). Biopsies were taken before (day 1) and after (day 15) treatment. Tumour volume was
defined as the sum of the longest diameters of all calliper- measurable mammary lumps (RECIST)
and tumour size as the longest diameter of the larger tumour (when multiple lesions). Both were
registered on days 1 and 15. ClinR was defined as a reduction in tumour volume or size at day 15
higher than 20%. The expression of progesterone receptors (PR) was analyzed by
immunohistochemistry in day 1 biopsies.

Results
No ClinR was registered in treated animals irrespective of the parameter (volume, size) employed.
PR-positive tumours (6 controls, 29 experimental) had a significant reduction in tumour size
(p<0.05) but not in tumour volume.

Conclusions
The heterogeneity of multiple synchronous canine mammary tumours with respect to histologic
type and PR status may be responsible for the lack of ClinR to aglepristone when the tumour
volume is measured. We propose the measurement of the longest diameter of the largest tumour
exclusively to evaluate ClinR of canine mammary carcinoma in the neoadjuvant setting.

Supported by PAIDI BIO287, P07-CVI-02559 and AGL2011-25553

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Ovariohysterectomy at the time of tumor removal decreases the risk of new tumors in dogs with
benign mammary tumors

Veronica Kristiansen
(Veronica Kristiansen, Ane Nødtvedt, Anne Marie Breen, Marianne Langeland, Thora Jonasdottir, et
al)

Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, Oslo,
Norway

[email protected]

Introduction
Benign mammary tumors (BMT) are common in intact female dogs. Little is known about their
clinical significance, and the effect of ovariohysterectomy (OHE). The purpose of this study was to
analyze new tumor development and cause of death in dogs with BMT, and identify factors
associated with risk of new tumors in a randomized clinical trial.

Materials
Dogs were randomly allocated to undergo OHE or not at the time of BMT removal. Benign tumor
was confirmed by biopsy in all subjects. Information about new tumor development was collected
via follow-up phone calls and recheck examinations. Two separate survival analyses were
performed with the endpoints new tumor development and death, respectively. Cause of death
was classified as related or unrelated to new tumor. Variables included age, tumor size, tumor
number, tumor duration, type of surgery, body weight, and OHE/intact status.

Results
83 dogs with 216 BMT were included (mean age 8.5 years). New tumor(s) developed in 24 of 42
(57%) intact dogs and 13 of 41 (32%) ovariohysterectomized dogs (hazard ratio 0.48, p=0.036). Nine
dogs (11%) were euthanized due to new mammary tumors. The risk of euthanasia related to new
tumors was not significantly different between the two treatment groups; in the intact group, five
dogs were subsequently treated for pyometra and two for uterine tumors.

Conclusions
OHE reduced the risk of new tumors by about 50% among bitches with BMT. Continued
endogenous hormonal exposure seems to increase the risk of additional mammary tumor
development in adult bitches.
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2 WVCC 2012 - 81 –

EVALUATION OF PAX-5 IMMUNOSTAINING AS A PAN B CELL MARKER IN PARAFFIN SECTION OF

FELINE LYMPHOMA: 31 CASES

Joaquim Henriques
(Joaquim Henriques, Ricardo Felisberto, João Matos and José Cabeçadas)

CICV- Faculty Veterinary Medicine, Lusofona University, Lisbon, Portugal

[email protected]

Introduction
Pax5 is a member of the family of transcription factors (B-cell specific transcription factor) and is a
key regulator in the commitment of B-cells. The goal of this study was to retrospectively assess
whether Pax5 protein in feline lymphoid cells is immunohistochemically detectable and whether its
expression is valuable for B-cell lineage determination.

Materials
Formalin-fixed paraffin-embedded tissues from 31 feline lymphomas were used. One reactive
lymph node with follicular reactive hyperplasia and one normal lymph node served as controls. For
immunohistochemistry a 3?m thick slides were stained with routine hematoxilin-eosin and further
immunophenotyping using validated antibodies anti- CD3 anti- CD79a and anti- Pax-5 was
performed.

Results
Of all B-cell lymphomas (18/31), 61,1% were determined by simultaneous expression of CD79a and
Pax-5; 38.9% were classified based on only one B cell marker: CD79a and Pax5 protein with 27.8%
and 11.1% respectively; In total, positivity for CD79 was seen in 88.9% of all B cell tumors. Thirteen
CD3 positive lymphomas were negative for anti-Pax5 and anti- CD79a staining, indicating a T-cell
lineage.

Conclusions
Anti-Pax5 antibody revealed inferior capacity to detect B cell tumors compared with CD79a. Co-
expression of Pax5 and CD79a was not evident in all B cell lymphomas, as it was described
previously for dogs. Probably this difference can be due to loss of the transcription factor in feline
lymphoma, or the discordant tumors being from neoplastic cells in a later maturation stage.
Despite these results, PAX5 should be considered as a valuable tool to diagnose B cell lymphoma in
cats.
 n d
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What spontaneous canine tumors tell us on the roles of the gap junction proteins, the connexins,
in cancer?

Maria Lucia Zaidan Dagli

(Maria Lucia Zaidan Dagli , Daniel Soares Sanches, Ivone Izabel Mackowiak Fonseca, Tarso Felipe
Teixeira, Emerson Flavio F Mota, Juliana Mariotti Guerra, Lucas Martins Chaible and Heidge
Fukumasu)

School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil

[email protected]

Introduction
Connexins are proteins that form the communicating gap junctions. Because of the importance of
intercellular junctions in the maintenance of the cellular homeostasis, their modulation seems to be
involved in carcinogenesis and cancer behavior. The aim of this report is to present evidences that
the study of connexins in spontaneous canine tumors may help to understand their roles in cancer.

Materials
Studies of the expression and subcellular localization of connexins in spontaneous canine mammary
tumors, bone tumors, oral melanomas, mast cell tumors and perianal gland tumors were
performed. Tumors were obtained from the Animal Pathology Service of the School of Veterinary
Medicine and Animal Science of the University Of São Paulo, Brazil. After histopathological
diagnosis and subtyping, tumors were submitted to the immunofluorescence staining for
connexins, and to other molecular techniques like Real-Time PCR and Western Blot.

Results
In all those spontaneous tumors, it has been verified that, in general, there is no decreased
expression of connexins in malignant tumors; however, a frequent finding was an aberrant
localization of these proteins in the cytoplasm, with decreased connexin spots in cell membranes.
These findings could be also related to the higher aggressiveness of the tumor subtypes.

Conclusions
In conclusion, the studied spontaneous canine tumors have shown an important role of connexins
in tumor behavior, which is similar to some human and experimental tumors. Studies of the causes
of this aberrant localization, its relationship with adhesion molecules like e-cadherin, and
possibilities to restore the gap junction communicating capacity in tumor cells, are ongoing.
 n d
2 WVCC 2012 - 83 –

Population-based incidence of spontaneous tumours in dogs living in Venice and Vicenza

provinces (north-eastern Italy)

Marta Vascellari
(Marta Vascellari, Katia Capello, Laura Bortolotti, Manuela Lanari, Antonio Carminato, Franco
Mutinelli and Elisa Baioni)

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy

[email protected]

Introduction
Cancer registration is considered key to any cancer control and prevention strategy. A well
designed and comprehensive population-based cancer registry is the most desirable from an
epidemiological standpoint, because it aims to represent all cases in a known population. In April
2005, a population-based tumour registry of dogs living in Venice and Vicenza provinces (Italy) was
established, to estimate incidence of spontaneous tumours in the study area.

Materials
The capture-recapture method with the Lincoln–Petersen formula was used to estimate the canine
population. Two independent sources were considered: the official regional canine demographic
registry (capture) and data from a telephone survey, performed on a random sample of 2,460
families, stratified by provinces (recapture). Incidence rates (IRs) were calculated as the annual rate
per 100,000 dogs, in the study period (72 months); for each IR an uncertainty range based on the CI
of the population size, has been reported,

Results
Overall, 226,801 dogs living in Venice and Vicenza provinces (95%CI: 211,473-246,889) were
estimated, and 5,866 tumours were registered, accounting for an IR of 431.1 (395.9-462.3), equally
distributed between malignant and benign tumours. The most represented cancers were mammary
tumours (IR=81; 74-87), mast cell tumours (IR=37; 34-39), testicular tumours (IR=20; 18-21),
lymphoma (IR=9; 8-10) and melanoma (IR=7.6; 7.0-8.1).

Conclusions
This study provides an accurate estimation of the canine population in the study area, as well as
epidemiological data about spontaneous tumour incidence in dogs. Information from cancer
registries can support studies of cause and prevention, as well as diagnosis, and survival of different
tumour entities.
 n d
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Creation of a xenotransplantable model for the study of canine mast cell tumors.

Marcia Kazumi Nagamine

(Marcia Kazumi Nagamine, Katia Cristina Pinello-Derneka, Claudia M C Mori, Katia Cristina Kimura,
Daniel Soares Sanches, Luciana Neves Torres and Maria LZ Dagli)

School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil

[email protected]

Introduction
The mast cell tumor is one of the most common neoplasms of the canine skin. Although much
progress has been achieved for the treatment of these tumors, novel studies are still required for
their better control. For this purpose, a xenotransplantable model for the study of MCTs has been
standardized. This paper describes the establishment and characterization of a canine
xenotransplantable model of mastocytoma model for testing substances with anticancer potential
in vitro and in vivo.

Materials
The tumor sample was original from a donor animal that showed a grade 3 mast cell tumor. The
tumor was digested with collagenase and immediately transplanted to athymic BALB / c nu / nu
mice. A part of this tumor was kept in primary culture, and reseeded several times.

Results
The canine mast cell tumor was successfully established in athymic mice BALB / c nu / nu. The
tumor has same histological characteristics retained during their passages in culture. On average, 7
days to 3 weeks are needed for the appearance of a palpable mass and about 60 days for tumor
volume reaching more than 500 mm3. Cultivation of canine mastocytoma cells is maintained for
approximately one month, sometimes more, but the cultivation time is limited. There is progressive
loss of the initial features of culture such as loss of granulation, increased adhesion and decreased
cell suspensions

Conclusions
These data show that the established model of murine xenotransplantable mastocytoma is stable
and viable and has importance for in vitro and in vivo tests with antineoplastic drugs.
 n d
2 WVCC 2012 - 85 –

Immunohistochemical characterization and EGFR expression of canine thymic tumors

Giuliano Bettini
(Giuliano Bettini, Maria Morini, Silvia Sabattini and Laura Marconato)

University of Bologna - Department of Veterinary Medical Sciences, Italy

[email protected]

Introduction
Thymoma is the most common primary mediastinal tumor in dogs; although derived from thymic
epithelial cells, it often displays prominent lymphocytic infiltration (lymphocytic thymoma), which
makes lymphoma its main differential diagnosis. Conversely, epithelial thymomas are
morphologically undistinguishable from epithelial tumors in other organs; thymofibrolipomas are
composed of cords of epithelial thymic cells admixed with fibroadipose tissue. The
immunohistochemical profile of canine thymic tumors has been poorly investigated, and
pathological markers for prognosis are also lacking.

Materials
Immunohistochemistry was carried out on canine samples from 12 thymic tumors and 5 normal
thymus, aiming at verifying and quantifying the expression of lymphoid markers (CD3, CD79),
cytokeratins (CKAE1/AE3, CK5/6, CK14, CK19), vimentin, TTF-1, COX-2, tyrosine-kinase-receptors
(EGFR and KIT) and Ki67.

Results
There were 6 lymphocytic thymomas, 4 epithelial thymomas and 2 thymofibrolipomas; tumor size
varied from 3 to 20 cm, and survival time after diagnosis ranged from one month to two years
(mean 5 months). Three dogs underwent radiation therapy and one surgery. Infiltrating
lymphocytes were mostly CD3+; thymic epithelial cells constantly and strongly expressed
CKAE1/AE3 and CK19, while COX-2 and EGFR were expressed at a variable degree; KIT was labeled
in sparse infiltrating mast cells and in one epithelial thymoma; Ki67 positivity was detected in 5-70%
of epithelial cells and in 5-50% of lymphoid cells. There was no evident association between these
markers and biological behavior.

Conclusions
Cytokeratin immunolabeling revealed useful in detecting epithelial cells in lymphocytic thymomas.
Although not directly related to the tumor behavior, EGFR expression suggests a possible indication
for targeted therapy.
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Effect of oncolytic vaccinia virus on malignant canine and feline cell lines in vitro and in vivo

Karoliina Autio
(Karoliina Autio, Marko Ahonen, Akseli Hemminki, Anna Knuuttila and Anja Kipar)

Department of Equine and Small Animal Disease, Faculty of Veterinary Medicine, University of
Helsinki, Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, Finland

[email protected]

Introduction
Cancer is one of the most common reasons for death in dogs, cats and humans. New therapeutic
modalities are necessary to improve the disease outcome. One promising approach is oncolytic
virotherapy based on replicating viruses. We assessed the oncolytic effect of double deleted
vaccinia viruses (vvdd) in vitro and in tumour-bearing mice.

Materials
Assays were based on two canine osteosarcoma and one prostatic carcinoma, and one feline
squamous cell carcinoma cell line. Luciferase assay was used to assess viral transduction in vitro.
Cell viability after viral infection was measured using a colourimetric cell lysis test. Nude NMRI mice
served as in vivo model and received a subcutaneous injection of canine prostatic carcinoma cells in
both flanks. They also served to test the vaccinia virus for pathogenic effects in organs. Mice were
treated twice with the vvdd or placebo at 10-day intervals.

Results
Infection resulted in efficient viral transduction and cell killing effect, with evidence of viral
inclusion body formation and increased cell death. In tumour-free nude mice, intravenous viral
infection was shown to induce viraemia but did not lead to any pathological changes. In xenograft
bearing mice, virus treatment lead to reduction in tumour size and histological evidence of viral
infection of neoplastic cells.

Conclusions
Oncolytic vaccinia virus has an anti-tumour effect on selected canine and feline cell lines in vitro
and a canine prostatic carcinoma cell line in vivo. Based on these data, further biodistribution and
toxicity studies will be performed in experimental dogs ultimately enabling the design of a clinical
trial.
 n d
2 WVCC 2012 - 87 –

Testing of antineoplastic properties of natural products in a xenotransplantable model of canine

mast cell tumor.

Marcia Kazumi Nagamine

(Marcia Kazumi Nagamine, Daniel Soares Sanches, Andréia Oliveira Latorre, Tereza Cristina da Silva,
Katia Cristina Kimura, Gregory Mennecier, Heidge Fukumasu and Maria LZ Dagli)

School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil

[email protected]

Introduction
A xenotransplantable model of a canine grade 3 mast cell tumor (MCT) has been standardized, and
natural products were tested for antineoplastic properties

Materials
The following doses of the natural products were tested: trichostatin A (TSA, metabolic product of
the fungus Streptomyces sp in vitro doses of 100 to 400 nM and in vivo dose of 100 µl/animal of
0,5mg/kg) and butanolic residue of the Pfaffia paniculata (RBPP) (Brazilian ginseng in vitro doses of
100 to 1000 µg/ml and intratumoral dose of 150 µl of 1 mg/ml).

Results
The TSA and RBPP showed inhibitory effects on the canine mastocytoma cells in vitro with a
decrease in cell viability detected with the vital dye Trypan blue and a decrease in cell growth
assessed by the MTT assay. The evaluation of the cell cycle measured by flow cytometry showed an
increase of cells in sub-G1 phase in cells treated with TSA, and a decrease in both G1 and G2 phases
and increased sub-G1 and S in the treatment RBPP. Despite the remarkable inhibitory effect of TSA
in vitro, it did not happen in vivo with the doses studied. RBPP showed an inhibitory effect with the
dosage of 1.5 mg / animal treatment in the in vivo model.

Conclusions
These data show that the established model of murine xenotransplantable mastocytoma is stable
and viable and has importance when in vitro and in vivo tests with antineoplastic drugs are
performed.
 n d
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The spatial distribution of Non-Hodgkin's Lymphoma in Humans and Dogs in the City of Sao
Paulo, Brazi: It's fact or coincidence?

Katia Kimura
(Katia Kimura, Zorelo Laporta, Fernanda Alessandra S Michels, Danielle Almeida Zanini, Adriana
Tomoko Nishiya, et al)

Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Brazil

[email protected]

Introduction
Non-Hodgkin's Lymphoma (NHL) shares similar morphological features, immunophenotype,
genotypic, clinic and prognostic characteristics in humans and canine species. We reasoned that, in
the case of spatial coincidence of NHL rates between two species, we would provide basis for the
understanding and quantification of the environmental role in the pathogenesis of NHL, since dogs
share with humans most of environmental and living conditions.

Materials
In the period of 1996-2006, 630 NHL lymphoma cases in humans were randomly selected among
more than 8000 cases at Cancer Registry of São Paulo; 579 canine NHL diagnosed in five centers
were also retrieved. The cases were geocoded by using the postal code of the person or of the dog
owner, and analyzed by software ESRI and the Arc MapTM 9.1 program.

Results
The spatial distribution of the cases of NHL human and canine in different districts of the São Paulo
municipality. An application of Spearman's correlation matrix and autoregressive error model
estimation demonstrated positive correlations between NHL human and canine clusters in three
scales. In this study, it was demonstrated higher concentration density in human and canine
lymphoma cases in the central region of São Paulo city where there was a lower population density
in both species and higher traffic density.

Conclusions
Spatial coincidence of NHL cases in both species may indicate common risk factors going beyond
the commonly investigated causes related to lymphoma subtype,suggesting a more detailed
investigation of environmental factors particularly associated with vehicle traffic, a major problem
in big cities.
 n d
2 WVCC 2012 - 89 –

The Development of a CSF-1 Receptor Monoclonal Antibody and Isolation of Canine Macrophages

Breno Castello Branco Beirão

(Breno Castello Branco Beirão, Lisa Yan-Toi Pang, David Hume and David John Argyle

University of Edinburgh - Roslin Institute, Edinburgh, UK

[email protected]

Introduction
Colony-stimulating factor-1 (CSF-1) is crucial for the maturation of cells of the mononuclear
phagocytic system. In many cancers, CSF-1 and its receptor (CSF-1R) are upregulated, leading to
infiltration of the tumor by macrophages. This is associated with poor prognosis. Therefore,
inhibition of the CSF-1 signaling pathway may be clinically relevant. We aim to raise an antibody
against the canine CSF-1R, to further understand the biology of dog macrophages and as a potential
therapeutic.

Materials
RNA was isolated from dog’s liver, from which CSF-1R cDNA was synthesized and cloned. To
perform microarray studies on dog macrophages, bone marrow was collected from dog’s femurs,
and macrophages were grown using human recombinant (hr)-CSF-1. To confirm isolation of
macrophages, FACS was performed based on the expression of CD11b.

Results
Macrophages were grown using hr-CSF-1. 96.4% of canine bone marrow derived cells grew into
macrophages, as assessed by CD11b positivity by FACS. We intend to determine the global gene
expression profile of canine macrophages. CSF-1R is a highly conserved, evolutionarily important
protein. Sequence alignment of canine, human and mouse CSF-1R show a level of high identity
between the species; 85% between human and canine and 80% between canine and murine. We
have begun successfully cloning canine CSF-1R. The extracellular portion of the cDNA was amplified
by PCR and cloned for expression of the protein.

Conclusions
Macrophages have been successfully produced from canine bone marrow and we have begun
cloning canine CSF-1R, with the intention of producing a monoclonal antibody as a therapy against
tumor-associated macrophages.
 n d
- 90 - 2 WVCC 2012

c-Kit immunostaining differs in canine buccal melanotic and amelanotic melanomas.

Tarso Felipe Teixeira

(Tarso Felipe Teixeira and Maria Lucia Zaidan Dagli)

Faculdade de Medicina Veterinaria e Zootecnia Universidade de São Paulo, Brazil

[email protected]

Introduction
Melanomas account 7% of all malignant canine tumors. They were classified in 3 subtypes
according to morphological aspects: epithelioid, spindle and mixed. Another classification is based
on the melanin expression: melanotic and amelanotic . The aim of this study was to evaluate c-kit
immunostaining in melanotic and amelanotic canine buccal melanomas.

Materials
A total of 37 melanomas from the buccal cavity of dogs were collected, being 20 melanotic and 17
amelanotic. We used HMB45 and Melan-A antibodies to differentiate the amelanotic melanoma
from other tumors. Immunohistochemistry to c-Kit was used to stain the melanomas. The number
and intensity of the immunolabeled cells was quantified in each sample, and then a group mean
was obtained. Statistical analysis was performed by Student t test.

Results
C-Kit protein was detected in the cytoplasm of 12/20 (60%) melanotic and in 10/17 (58.8%)
amelanotic melanomas. Melanotic melanomas showed that 58,3% of the cells were weakly stained,
25% of the cells were moderately stained and 16.7% of the cells were strongly stained. Amelanotic
melanomas showed that 10% of the cells were weakly stained, 30% of the cells moderately stained,
and 60% of the cells were strongly stained.

Conclusions
These results show that melanoma cells differ in the immunostaining, and that most amelanotic
melanomas presented a strong immunostaining for c-kit. These results may have importance when
considering the possible treatment of buccal melanomas with tyrosine-kinase inhibitors.
 n d
2 WVCC 2012 - 91 –

E-cadherin in canine mast cell tumours: decreased expression and altered subcellular localisation
in grade 3 tumours

Ivone Izabel Mackowiak da Fonseca

(Ivone Izabel Mackowiak da Fonseca, Luciana Boffoni Gentile, Lucas Martins Chaible , Marcia
Kazumi Nagamine, Juliana Mariotti Guerra, et al)

University of São Paulo, School of Veterinary Medicine and Animal Science, Brazil

[email protected]

Introduction
Mast cell tumours are the most frequent round cell tumours in dogs. These tumours are highly
invasive and metastatic in correspondence with the histological grade. E-cadherin is an adhesion
molecule expressed in epithelial cells. Although E-cadherin is an epithelial cellular marker, previous
studies have shown expression of E-cadherin in canine round cell tumours. To better characterize
the expression pattern of E-cadherin in several different histological grades of mast cell tumours in
dogs, we sought to study the expression and localization of the adhesion molecule by
immunohistochemistry.

Materials
For this purpose, 18 cutaneous mast cell tumours, were obtained, and classified into the three
histological grades of 1, 2 or 3 (6 cases of each). Clinical history and follow-up data were available
for all of the dogs. The tumores were immunostained for E-cadherine by using 2 different
antibodies: clone HECD1 and NHC38. Both recognize the extracellular portion of E-cadherine. The
tumor immunostainings were evaluated acoording to intensity and subcellular localisation.

Results
It has been verified cytoplasmic and nuclear expression of E-cadherin in all three types of tumours
by immunostaining using two different antibodies. This expression decreased in the more
aggressive mast cell tumours (grade 3), suggesting a correlation of E-cadherin with the tumour
aggressiveness.

Conclusions
This study demonstrates the expression of a characteristic epithelial marker in a mesenchymal cell
type, the mast cell. In addition, the loss of E-cadherin expression in either the cytoplasmic
membrane or nucleus in the more aggressive and undifferentiated tumour types highlights the
importance of cellular adhesion in tumour behavior.
 n d
- 92 - 2 WVCC 2012

Diagnostic difficulties of thyroid nodules and tumours in dogs and cats.

Julianna Thuróczy
(Julianna Thuróczy, Edina Perge, Linda Müller, Eszter Kollár and Lajos Balogh)

SzIU, Faculty of Veterinary Science Budapest, Hungary

[email protected]

Introduction
Aim of our study was to find correlation among results of histopathologic diagnosis, thyroid
hormone concentrations and pertechnetate scintigraphy of thyroid nodules and tumours.

Materials
Serum thyroxin (T4), thyrotropin (TSH) concentrations of thirty one dogs (16 females, 15 males) and
ten cats (5 females and 5 males) were measured parallel with technetium-pertechnetate
scintigraphy. Fourteen (45%) dog patients suffer from malignant and seventeen (55%) patients
suffer from benign thyroid transformations. Proportion in cats was 4 (40%) and 6 (60%).

Results
Serum TSH concentrations of eleven dog patients was lower and TSH of ten dog patients was higher
than physiological level. Serum T4 concentrations did not differ statistically in malignant and benign
tumours and species. Extremely high serum T4 concentrations were measured in cases of
multinodular hyperplasia, nodular atrophy and thyroiditis. In contrast, pertechnetate-uptake was
the highest in cases of compact cell carcinomas, follicular adenocarcinoma and follicular compact
cell carcinoma. Standard deviation of serum TSH concentrations was the highest in case of follicular
adenomas.

Conclusions
There was no statistical difference in pertechnetate-uptake between malignant and benign
tumours. There was no correlation between T4 and pertechnetate-uptake ranging on basis of
species or histopathology.
 n d
2 WVCC 2012 - 93 –

Influence of Hypothyroidism on Experimental Melanoma Progression

Luiz Roberto Biondi

(Luiz Roberto Biondi, Maria Lucia Zaidan Dagli, Fernanda Stachicini, Fabiana Aliperti and Mario
Mariano)

School of Veterinary Medicine and Animal Science University of Sao Paulo, Brazil

[email protected]

Introduction
Malignant melanoma is a life-threatening disease characterized by a highly aggressive biologic
behavior in both humans and dogs. Some authors have attempted to access the interaction
between cancer and thyroidal conditions, with controversial findings. This study aimed to verify the
melanoma B16F10 progression in mice submitted to hypothyroidism.

Materials
Forty 60-day-old male C57BL6 mice were divided in local growth evaluation with propylthiouracil-
PTU-induced hypothyroidism and metastatic evaluation with PTU and Iodine-131-induced
hypothyroidism. Control groups received tap water. Local groups received subcutaneously 1x106
B16F10 viable cells. Survival and tumor growth were followed daily and tumor measurement
performed with precision caliper. Metastatic groups received intravenously 2x105 viable B16F10
cells. Fifteen days after inoculation, both treated and non-treated mice were euthanized, their
lungs were removed and the number of nodes on the pulmonary surface counted through a
stereoscopic magnifying glass. Statistical analyses were performed trough unpaired T test and
ANOVA.

Results
The mean survival rate was 22 days on tumor growth assay. Log-rank test showed no significant
difference between groups. The number of metastatic nodules on the lung surface was 32 on
control group, 91 nodules on PTU group and 77 on I131group with statistically significant difference
(p<0.01 CI 99%) between group control and treated with PTU or I131.

Conclusions
In this trial, hypothyroidism did not influence the local growth of B16F10 melanoma; however,
hypothyroidism significantly altered the melanoma progression increasing the formation of
pulmonary nodules in C57BL6 mice in which hypothyroidism was induced either by PTU or I131.
 n d
- 94 - 2 WVCC 2012

ALPHA-1-ACID GLYCOPROTEIN CONCENTRATIONS IN CATS WITH LYMPHOMA UNDERGOING

CHEMOTHERAPY

Valter de Medeiros Winkel

(Valter de Medeiros Winkel, Samantha Ive Miyashiro and Sílvia Regina Ricci Lucas)

School of Veterinary Medicine and Animal Science-University of São Paulo, Brazil

[email protected]

Introduction
Alpha-1-acid glycoprotein (AGP) is an acute-phase serum protein that is produced by the liver in
response to inflammation, infection and neoplastic conditions. Lymphomas comprise one of the
most common groups of tumors in cats. The aim of this study was to determine AGP concentration
during chemotherapy and its relation with remission in cats with lymphoma.

Materials
AGP was measured by the use of a commercial species-specific immunoassay test (Tridelta LTD,
Ireland) in two groups of cats: 13 healthy cats and 16 cats with lymphoma, that had not received
any treatment prior the diagnosis. For both groups, serum samples were collected, harvested and
frozen at -70oC until assayed. Of the 16 cats with lymphoma, only 11 cats that presented remission,
were followed during chemotherapy, and evaluated at 2-week intervals along 8 weeks. Mann-
Whitney test followed by multiple Tukey´s test were used to compare the groups and weeks of
treatment.

Results
Mean AGP concentration was significantly higher ( p<0,0001) in cats with lymphoma at diagnosis
(1.237,9 +/- 815,81µg/mL) than in healthy cats (162,38 +/- 124,64µg/mL). Mean of AGP
concentration decreased gradually during the treatment, and it was statistically different at the 8th
week (452,79 +/- 201,44µg/mL) ( p<0,0235), when lymphoma remission was achieved and the cats
presented good clinical status.

Conclusions
In conclusion, AGP concentration was higher in cats with lymphoma prior to chemotherapy and
decreased gradually along the treatment when remission is achieved, but did not return to normal
values.
 n d
2 WVCC 2012 - 95 –

Changes in positron emission tomography (PET) uptake of 18F-fluorodeoxyglucose (18FDG), 18F-

fluoro-3’-deoxy-3’-L-fluorothymidine (18FLT) and Copper(II)diacetyl-bis(N4-methylthiosemi-
carbazone) (64Cu-ATSM) before, during and after radiotherapy of a canine

Kamilla Westarp Zornhagen

(Kamilla Westarp Zornhagen, Malene Martini Clausen, Fintan J McEvoy, Annemarie T Kristensen,
Anders E Hansen, et al)

Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen,
Denmark

[email protected]

Introduction
Non-invasive functional imaging of tumour glycolysis, hypoxia and proliferation using combined
PET/CT can potentially serve as a basis and evaluation parameter for radiotherapy. The aim of this
study was to investigate PET uptake of 18FLT (proliferation), 18FDG (glycolysis) and 64Cu-ATSM
(hypoxia) before, during and after radiotherapy.

Materials
Mixed breed dog (9y/FS) with a tibial fibrosarcoma received 10 fractions of 4.5Gy radiotherapy.
18FLT, 18FDG and 64Cu-ATSM PET/CT scans were performed, before, during, at completion of and
10 weeks after radiotherapy. Images were co-registered and the tumour-region delineated on the
18FDG PET/CT scan (True X reconstruction) using 3D isocontouring in True D (SUV cut off 2.5).
Regions were applied to the corresponding 18FLT and 64Cu-ATSM images. Tumour SUV maximum
and mean uptake of all tracers was subsequently determined.

Results
The initial uptake of 18FDG decreased during therapy and maximum uptake was reduced by
&gt;60% after therapy. 18FLT uptake displayed fluctuations, but maximum uptake was reduced by
&gt;65% after therapy. 64Cu-ATSM uptake decreased continuously during the course of
radiotherapy. Visual interpretations of the PET/CT scans also reflect the decrease in tracer uptake.

Conclusions
The use of hypofractionated radiotherapy protocols is currently subject for debate. 10 fractions of
4.5Gy radiotherapy was very well tolerated, however late side-effect cannot yet be evaluated.
18FLT fluctuations could be associated with accelerated repopulation occurring during therapy. The
decline in 64Cu-ATSM indicates that tumour reoxygenation occurred. The continued, but reduced,
uptake of 18FDG after radiotherapy could illustrate incomplete tumour control, but may also to
some extent be associated with tissue remodelling.
 n d
- 96 - 2 WVCC 2012

Relationship between IL-8 and Slug in proliferation and survival of canine hemangiosarcoma cells

Jong-Hyuk, Kim
(Jong-Hyuk~ Kim,
Aric M Frantz,
Ashley J Graef, TaeHyun Hwang, Milcah C Scott, Aaron L Sarver, Leslie C Sharkey, et al)

Masonic cancer center, University of Minnesota, Minneapolis 55455, USA

[email protected]

Introduction
Hemangiosarcoma (HSA) is a common, highly metastatic and incurable tumor in dogs. However, its
biological and pathological features are poorly understood, hindering development of effective
therapies. We showed previously that there is enhanced activity of a biological network centered
on interleukin-8 (IL-8) in canine osteosarcoma. IL-8 is a proinflammatory chemokine that is
important for progression and survival in other tumors. Our goal was to establish if IL-8 was
required for proliferation and survival of canine HSA.

Materials
We determined expression of IL-8 mRNA and protein in four HSA cell lines grown in the presence of
absence of nutrients (serum). RNA levels were quantified using real time RT-PCR, cell-associated
protein levels were assessed by immunoblotting, and secreted IL-8 was measured by ELISA. We also
examined correlations between IL-8 expression and canonical signaling pathways based on
genome-wide gene expression profiles in 12 HSA cell lines.

Results
Expression of IL-8 was variable among the HSA cell lines tested, and the cells did not consistently
respond to growth factor deprivation by upregulating IL-8. Intriguingly, expression of Slug, a Snail
gene family member involved in epithelial-mesenchymal transition and cell survival, was inversely
correlated with IL-8, specifically in cell lines derived from splenic HSAs.

Conclusions
The data suggest that IL-8 is not essential for proliferation and survival of all HSAs, and that at least
in splenic HSAs, this lack of reliance on IL-8 may be due to compensatory mechanisms that engage
alternative pathways such as those controlled by Slug to promote growth and survival.
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2 WVCC 2012 - 97 –

Expression of cytoskeletal proteins and adhesion molecules in three canine mammary cell lines.

Adelina Gama
(Adelina Gama, Natália Rocha, Daniela Pereira and Joana Paredes)

Laboratório de Histologia e Anatomia Patológica, Trás-os-Montes e Alto Douro University (UTAD) &
Veterinary and Animal Research Centre (CECAV), Portugal

[email protected]

Introduction
Canine mammary tumours are the most common neoplasia in female dogs, being characterized by
a high biological and morphological heterogeneity.

Materials
In this study, a characterization of three cell lines was performed: CMT-2p and CMT-3p, established
from primary mammary carcinomas, and CMT-1m, obtained from a metastasis of a canine
mammary carcinoma. We have assessed their morphology, growth kinetics and pattern of
expression to cytoskeletal proteins (cytokeratins and vimentin) and adhesion molecules (E-cadherin
and ß-catenin).

Results
Cell lines presented an epithelioid morphology and different growth kinetics. All cell lines showed
extensive positivity for cytokeratins, which confirmed their epithelial phenotype. However, two cell
lines showed co-expression of vimentin, which may indicate an epithelial-mesenchymal transition.
With regard to adhesion molecules, cell lines showed different expression patterns, from preserved
membrane to cytoplasmic expression, which may reflect distinct invasive capacities. In the case of
?-catenin, nuclear staining was also observed, which may be related to its possible involvement in
the activation of transcription factors.

Conclusions
The establishment and characterization of new canine mammary cell lines is important, since there
is still a rather small number of cell lines available, which might constitute a useful tool for the
study of canine mammary tumours, namely in the development of novel therapies.
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Isolation, identification, differentiation and radiolabelling of canine adipose tissue derived

mesenchymal stem cells (cAD-MSC)

Lajos Balogh
(Lajos Balogh, Veronika Haász, Zita Postenyi, Andras Polyak, Katalin Nemet, et al)

NRIRR Budapest, Hungary

[email protected]

Introduction
Our aim was to establish isolation-, differentiation- and radiolabelling method of canine adipose
tissue derived mesenchymal stem cells (cAD-MSC) and to prove the usefulness of the canine model
for human biomedical sciences.

Materials
The adipose tissue samples were collected from visceral and different subcutaneous fat depots
from Beagle dogs using standard sterile surgical procedures. The SVF (Stromal Vascular Fraction)
was obtained by digestion with collagenase then after centrifugation cells were incubated in
Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).
Cells were successfully differentiated towards adipogenic, osteogenic and chondrogenic lineages.
Moreover, FACS measurements were carried out to identify the expression of the appropriate cell
surface markers. After radiolabelling (99mTc-HMPAO, Leuco-Scint® kit) the distribution of
reinjected stem cells were imaged by SPECT/CT technique.

Results
The adipose derived MSC cells, similarly to the human adipose derived cells, showed fibroblast-like
morphology in light microscope. The phenotype of the isolated cAD-MSC was identified by
detecting cell surface markers with flow cytometry (FACS); that is we successfully isolated canine
adipose derived stem cells. Non-specific radiolabelling with 99mTc-HMPAO resulted high labelling
efficiency with retained functional abilities so that intravenously and intraarticularly reinjected
MSCs could be followed-up upto 24hours postinjection by SPECT/CT.

Conclusions
Our preliminary results suggest that isolation-, identification-, differentiation- and radiolabelling of
cAD-MSC is feasible. Canine adipose tissue represents an easily available source for veterinary stem
cell therapies.

Scientific work was supported by several national (OTKA-68376, JEDIONKO, KMOP-1.1.1-08/1-2008-

0017, GOP-1.1.1.-09/1-2010-0107) and international (IAEA-CRP, EMIL NoE) projects.
 n d
2 WVCC 2012 - 99 –

Canine Breast Cancer: Prevalence in Santos City, Sao Paulo-Brazil

Luiz Roberto Biondi

(Luiz Roberto Biondi, Maria Lucia Zaidan Dagli and Alexandre Mattos Rego)

School of Veterinary Medicine and Animal Science University of Sao Paulo, Brazil

[email protected]

Introduction
Mammary tumors are among the most common tumor in dogs, ranging from 25–50% of all
neoplasms. Actually, estimates of annual incidence of canine mammary neoplasia (CMN) depend
on the population studied and vary widely from 198 per 100,000 to 111 per 10,000 female dogs,
correlated to early spaying. Because CMN may serve as relevant model for human breast cancer,
there is an increasing interest in the knowledge of its incidence and histopathological classification.

Materials
Data of 14,298 dogs were retrospectively collected from medical records of Veterinary School of
Santos Metropolitan University, from a period of 72, searching for histopathological diagnosis of
cancer.

Results
From 7882 males and 6416 females, 505 histopathologic diagnoses of tumors were obtained,
representing 3.6% of this population, distributed in 74 benign tumors and 431 malignant tumors.
Among females, 37 tumors were benign and 280 malignant, of which 174 mammary tumors,
equivalent to 54.9% of this diagnosis in dogs. The benign breast tumors diagnosed were 8 simple
and 6 complex adenomas, corresponding to 4,4% of all breast tumors and the malignant were 99
simple tubular carcinomas, 44 complex carcinomas, 8 ductal carcinomas, 8 solid carcinomas and 1
carcinoma-malignant myoepithelioma, accounting for 95,6% of cases. Within the group of breast
tumor diagnosis, 19 females had a history of spaying prior the tumor diagnosis, corresponding to
10.9%.

Conclusions
The high prevalence of spontaneous malignant breast cancer in female dogs of City of Santos due
to the large number of non-spayed animals in this population, represents valuable material for
comparative studies.
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MALDI mass spectrometry (MALDI-MS) imaging of lipids in canine mammary tumor sections

Michele S. Frehse
(Michele S. Frehse, Roberta Lemos Freire, Ana Paula R F L Bracarense, Elisângela Olegário da Silva,
Fernanda Romero, et al)

Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina - UEL,PR,

Brazil

[email protected]

Introduction
Mammary neoplasms are the most common neoplasm in female dogs. They represent 50% of the
tumors in bitches and half of them are diagnosed as malignant. The aim of this study was to
perform MALDI-MS imaging of lipids species present in mammary tumors and compare their
presence and distribution with normal tissue samples.

Materials
Six female dogs (3 poodle, 1 cocker spaniel, 1 boxer, 1 mongrel) mean 12-yr-old were submitted to
mastectomy at the Veterinary Teaching Hospital-Universidade Estadual de Londrina (UEL).
Histopathology revealed 1 Carcinoma grade I; 2 Carcinoma grade II; 1 Hemangiopericitoma; 1
Carcinosarcoma and 1 Mixed benign tumor. Normal breast tissues from 3 control (cadaver) also
tested. For MALDI-MS imaging, tissue samples were sliced (20µm), placed directly in the target
plates and covered with 2,5-dihydroxy benzoic acid (DHB) 0.5M. Experiments were performed in an
Autoflex III mass spectrometer in the positive ion mode at the m/z range of 600-1200.

Results
Lipid species observed were mostly structural lipids (sphyngomyelins and phosphocholines) and
different distributions of some lipid ions (m/z 782.6, 760.6) were observed between cancerous and
normal tissues. Currently the structural characterization of the lipid species and increasing the
number of samples were analyzed in order to evaluate the perspectives of the technique.

Conclusions
As already observed for many types of cancer, MS imaging of lipids is able to provide cancer
diagnostic and tumor grading since it allows obtaining extensive chemical information and location.
We envisage the application of these technologies also for research and diagnostics of mammary
tumor in bitches.
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2 WVCC 2012 - 101 –

Masitinib in High Risk Mast Cell Tumors: a prospective, Phase 3, non-randomized clinical trial

Elsa Beck
(Elsa Beck, Conor McNeill and Bowers Tessa,)

The Hope Center, Vienna, USA

[email protected]

Introduction
Masitinib is a tyrosine kinase inhibitor that has been widely used to treat a variety of tumors,
especially mast cell tumors. Recently, mast cell tumors of a lower grade (grade 1 or 2 with low
mitotic index) have been shown to have low failure rates even with incomplete resection. By
constrast, high risk mast cell tumors (grade 3 or grade 2 with a high mitotic index) have continued
to be a challenge. They typically recur and metastasize with very few long term survivors.

Materials
Thirty four dogs with high risk mast cell tumors were treated with masitinib in a prospective,
nonrandomized, Phase 3 clinical trial. Some of these dogs had been heavily pretreated, but a short
washout time was required. Treatment was discontinued because of toxicity, rapidly progressive
disease, or owner request.

Results
38% of these dogs were continued on this medication for less than 4 weeks. The reason for
withdrawal was progressive disease in 50%, toxicity in 40% and financial constraints in 10%. The
mean and median treatment length was 2 weeks. Dogs that had been heavily pretreated were
more likely to be in this catagory. For the remaining dogs, the mean treatment length was 5
months, with a median of 2 mo. None of these dogs were withdrawn for toxicity reasons, 30% had
progressive disease, and the remaining 70% have remained disease free, several for longer than 6
months.

Conclusions
Masitinib is useful as a single agent in high risk patients. Treatment incombination with traditional
chemotherapy should be investigated.
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DOG BRAIN TUMOR RESECTION USING INTRAOPERATIVE MRI

Hiroshi Ishii
(Hiroshi Ishii, Akihide Wada, Shohei Kodera, Naohide Minami, Yoshiharu Okamoto, et al)

Tokyo Animal Medical Center, Tokyo, Japan

[email protected]

Introduction
Since brain tumors can hardly distinguished from normal brain tissue, trying to ensure that the
tumor is fully resected may result in sequelae, and, conversely, being overly conservative in
removing tissue leads to a reduced survival rate. We therefore conducted dog brain tumor
resections using intraoperative MRI, which we report here.

Materials
The dogs undergoing treatment were a 10-year-old male Shetland sheepdog with a mass in the left
lobe (Dog 1) and an 11-year-old female Welsh corgi with a mass in the right ventricle (Dog 2). The
surgery was performed in an intelligent animal operating theater equipped with state-of-the-art
medical devices and techniques such as an open MRI, a real time update navigation system, a
dissecting microscope and an operating table compatible with MRI. The surgery was performed
while monitoring the position of lesions with the navigation system, and the absence of
hemorrhaging and reminiscent tumors was confirmed with MRI before the end of surgery.

Results
The masses were resected almost completely from the two dogs undergoing the present surgical
procedure. The dogs recognized their guardians and were able to walk normally about 3 hours after
the closure of cranium. Dog 1 and Dog 2 were diagnosed as having malignant histiocytic sarcoma
and cavernous hemangioma, respectively, on the basis of the results of the postoperative
pathological examination.

Conclusions
The intelligent animal operating theater, in which imaging with intraoperative MRI is possible
whenever required without moving animals, appears to be an ideal operating system that enables
safe and precise resection of brain tumors.
 n d
2 WVCC 2012 - 103 –

SPONTANEOUS PROGNOSIS AND IMMUNOPHENOTYPE OF FELINE MAMMARY CARCINOMAS

Frédérique NGUYEN
(Frédérique NGUYEN, Catherine IBISCH, Lise BERTRAND, Mélanie POHU, Floriane MORIO, et al)

Oniris Dept. of Pathology, Nantes, France,

[email protected]

Introduction
We aim to establish which clinical, histological and immunohistochemical criteria have prognostic
significance in feline mammary carcinomas (FMCs) regarding time to progression (recurrence,
metastasis), overall survival, and specific survival.

Materials
Retrospective study of 289 invasive FMCs treated by surgery alone, with available 2-year follow-up.
Recorded data: breed, age, age at neutering, cause and time to death, tumor size, histological
subtype, Elston &amp; Ellis grade, mitotic index, emboli. Immunophenotype defined as in human
breast cancer using antibodies to: estrogen and progesterone receptors (ER, PR), Her-2,
cytokeratins 5/6, Epidermal Growth Factor Receptor, and Ki67.

Results
Age at diagnosis: 11.2±2.8 years. 50% intact, 50% neutered females. 56% of the tumors are more
than 20mm in diameter. Histologic subtypes: cribriform (52%), solid (23%), mucinous (12%), and
tubular/papillary (13%). Grades: III (51%), II (46%), I (3%). Emboli observed in 43% of FMCs. At the
end of the study, the recurrence rate is 32%, metastasis rate is 46%, cancer-related death rate is
73%. By multivariate analysis, factors predicting specific survival include tumor size, lymph node
metastasis, distant metastasis, recurrence, lymphatic emboli, histological grade, dermal infiltration.
80% of FMCs are ER-negative, 94% are PR-negative. Only 1 Her2-overexpressing FMC was found.

Conclusions
Invasive FMCs are aggressive neoplasms, with a median survival of 249 days. Most (76%) are triple
negative (ER and PR negative, not Her-2-overexpressing) carcinomas. This finding validates the cat
as a model to test new therapeutic strategies for human triple negative breast cancer.
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Modulation of Cytokines Production by PGE&#8322; in Ehrlich Ascites Tumor.

Luciana Boffoni Gentile

(Luciana Boffoni Gentile and Denise Fecchio)

Lab of Experimental and Comparative Oncology, School of Vet Medicine and Animal Science, USP,
Sao Paulo, Brazil

[email protected]

Introduction
The use of experimental models has contributed to evaluate the inflammatory response against
tumors. Ehrlich ascites tumor (EAT) has been used as a model to evaluate the inflammatory
response against the tumor growth due to the development in all mice strains and easy
manipulation.

Materials
Swiss mice were inoculated with 1x10^9; tumor cells (ip) and treated with indomethacin (1mg/Kg,
1x/day, ip). After 1, 3, 6, 10 and 13 days the animals were evaluated for secretion of IL-1α, IL-2, IL-4,
IL-6, IL-10, IL-13, TNF-α and PGE2 in peritoneal cavity by ELISA.

Results
The results have shown the inoculation of EAT cells induces PGE2 production during all neoplastic
evolution. EAT induces production of IL-6 from 10th day and of IL-2 on 13rd day. The indomethacin
treatment of EAT bearing mice inhibited the tumor growth and PGE2 synthesis from the 10th day.
Indomethacin treatment also promoted IL-13 synthesis and significant inhibition of IL-6 on 13rd day
of EAT growth.

Conclusions
In conclusion, the treatment with indomethacin was effective in inhibiting the growth of TAE. Also,
the evaluation of the cytokines profile in the treatment of TAE-bearing mice with indomethacin,
allowed us to infer that indomethacin positively modulates the production of IL-13; negatively the
release of IL-6 and does not alter the profiles of IL-1α, IL-2, IL-4, IL-10 and TNF-α.. PGE2 modulates
the growth of TAE and the inhibition of tumor growth could be partly related to suppression of the
IL-6 and IL-13 stimulation.
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2 WVCC 2012 - 105 –

THE USE OF LYMPHOSCINTIGRAPHY WITH CT-SCAN IMAGE FUSION TO DETECT SENTINEL LYMH
NODES IN DOGS WITH CANCER

LAURENT MARESCAUX
(LAURENT MARESCAUX, EMILIE CATHELAIN ,FRANÇOIS SERRES and DOMINIQUE TIERNY)

ONCOVET, Villeneuve d’Ascq, France

[email protected]

Introduction
The Sentinel Lymph Node (SLN) is described as the first lymph node receiving drainage from a
specific anatomic region. In humans, SLN identification and biopsy are critical in the staging and
surgical treatment planning of certain cancers such as breast cancer and melanoma.
Lymphoscintigraphy is one of the methods used for sentinel lymph node mapping.

Materials
Lymphoscintigraphy was performed in three dogs with recently excised tumors (one dog with
malignant mammary tumor, one dog with digital melanoma and one dog with palmar mast-cell
tumor). Two to 4 MBq of Tc-99m sulfur colloid per kilo was injected around the tumor site. Dogs
were under anaesthesia. Computed Tomography (CT) was performed before the
lymphoscintigraphy, in the same position.

Results
Lymphoscintigraphy revealed the SLN for the three dogs (one axillar and one prescapular lymph
node for the two dogs with distal forelimb tumors and one illiac lymph node for the dog with
mammary cancer). Images were superimposed with those of the CT to precise the anatomic
position of the lymph node.

Conclusions
Lymphoscintigraphy combined with CT imaging appears to be very useful in the detection of
sentinel lymph nodes in pets with cancer. Because of the variation in lymphatic drainage in both
human and animals, identifying and removing the SLN can likely provide more accurate staging,
help guide treatment and possibly influence prognosis. Prospective studies are needed to further
define the role of this technique in veterininary medicine.
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Growth hormone receptor (ghr) RNAi decreases proliferation and enhances apoptosis in CMT-
U27 canine mammary carcinoma cell Line

Karol Pawlowski
(Karol Pawlowski, Dominika Popielarz, Katarzyna Szyszko, Margorzata Gajewska, Tomasz Motyl and
Magdalena Król)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Department of Animal

Environment Biology, Faculty of Animal Sciences, Warsaw University of Life Sciences, Warsaw,
Poland

[email protected]

Introduction
High GH level may enhance risk of tumourigenesis. Based on our studies we suppose that high
proliferative and anti-apoptotic potential of canine mammary cancer cells was related to the up-
regulation of GHR. Hereby we evaluated the GHR role in the biology of canine mammary carcinoma
cell line

Materials
The canine mammary carcinoma cell line CMT-U27. Standard siRNA transfection procedure has
been outlined. The ghr silencing was confirmed using the realtime PCR and immunohistochemistry.
The Western blot analyses were conducted to confirm whether the level of GHR reduction is
correlated with any perturbations in downstream intracellular signalling cascade . We also anylised
cell cycle of transfected cells using Annexin V and propidium iodide .

Results
89% GHR gene silencing efficiency was reached. The RT-qPCR confirmed the effect of ghr gene
silencing (43.21% expression reduction compared against control cells) The immunohistochemical
studies revealed 64.87% efficacy of gene silencing at the protein level. The results of Western-blot
indicated that the reduction of GHR was followed by decrease in p-ERK1 and p-ERK2 expression
(49% and 47% decrease compared to control level, respectively) Analysis of apoptosis using
AnnexinV and PI showed that 30.8% the control cells were apoptotic whereas siRNA treatment
gave 42.3% apoptotic cells. 5.7% analyzed cells proved an early stage, whereas 25.05% a late stage
of apoptosis. Cells treated with ghr-specific siRNA showed 12.87% of early and 29.5% of late
apoptosis respectively.

Conclusions
Based on the results we propose that GHR signalling tilts the balance between the cellular control
of proliferation and death in CMT-U27.
 n d
2 WVCC 2012 - 107 –

Evaluation of pulmonary metastases by single breath-hold helical CT in cats with fibrosarcomas.

Pauline de Fornel-Thibaud
(Pauline de Fornel-Thibaud, Patrick Devauchelle and Françoise Delisle)

Centre de Cancérologie Vétérinaire, Paris, France

[email protected]

Introduction
Feline fibrosarcomas are highly recurrent tumors with low metastatic rate, estimated between 0 to
28% by radiography. The purpose of this study was to evaluate the prevalence of pulmonary
metastases in cats with fibrosarcomas and to describe the population with metastases.

Materials
CT studies performed between December 2009 and October 2011 on cats with fibrosarcomas were
reviewed. Inclusion criteria were: primary or recurrent fibrosarcoma in place or just removed;
pulmonary nodules detected on helical CT; no history or diagnosis of other tumor.

Results
The CT studies of 200 cats were reviewed: 73 with primary tumors and 127 with recurrent tumors.
Pulmonary nodules were detected in 29 cases. Four cases were excluded for previous or
concomitant malignant tumors. Finally 25 cats met the inclusion criteria: 11 males, 14 females,
median age 12 years (7-17 years). One pulmonary nodule was detected in six cats, between two
and five nodules in seven cats and more than five in the others. In six cats, the primary
fibrosarcoma was in place or just removed. The size of the tumors ranged between 6 to 240 cm3.
Metastases were found after the first recurrence in six cats, after the second recurrence in nine,
after the third recurrence in four and more for the last two cats. The median time from primary
surgery to detection of pulmonary metastases was 12 months (2-36 months).

Conclusions
These results confirm the low metastatic rate of feline fibrosarcomas, mainly associated with
recurrences. In few cases, metastases occurred on early recurrences and even on primary tumors.
 n d
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CORRELATION OF PRETREATMENT THYMIDINE KINASE CONCENTRATION WITH REMISSION

DURATION AND SURVIVAL IN DOGS WITH LYMPHOMA

Sara Westberg
(Sara Westberg, Kim A Selting and Henrik von Euler)

University Animal Hospital, Swedish University of Agricultural Sciences (SLU), Sweden

[email protected]

Introduction
Thymidine kinase (TK) is a soluble biomarker present in S-phase of a salvage pathway for DNA
synthesis, and can be measured in serum. TK activity correlates with stage, prognosis, and relapse
in dogs and humans with lymphoma. Unpublished data suggests that TK cannot distinguish
responders from non-responders. The purpose of this study was to correlate TK concentrations
among dogs with uncontrolled lymphoma with duration of response to standard chemotherapy,
considering other strong prognostic factors.

Materials
Pet dogs (n=67) presenting with lymphoma in Sweden and the US were fully staged when possible
and evaluated including TK assessment. TK values were recorded as were other known prognostic
factors (immunophenotype, blood calcium concentration, prior treatment with prednisone, and
type of chemotherapy used). Remission status within the first 4 weeks of initiation of
chemotherapy, duration of remission and overall survival were recorded and evaluated, using
Kaplan Meier analysis.

Results
All dogs included were treated with doxorubicin-based multidrug chemotherapy. Only 2 dogs had
received prednisone in the 3 weeks prior to assessment and chemotherapy. Five dogs were
hypercalcemic. TK concentrations ranged from 1.6-356 U/L (median 91.5 U/L, normal <6 U/L).
Overall survival was 7 months for dogs with TK concentration less than 40 U/L, and 9 months for
higher values (P=0.02). Advanced stage correlated with decreased chance of attaining remission
(P=0.005).

Conclusions
Though TK concentrations were variable in this multicontinental study, high TK was associated with
statistically shorter survival in this large cohort of dogs with lymphoma.
 n d
2 WVCC 2012 - 109 –

PHASE I/II CLINICAL TRIAL OF CTI52010 IN TUMOR-BEARING DOGS

Kim A. Selting
(Kim A Selting, Sandra M Axiak, Jeffrey N Bryan, Charles Decedue and Carolyn J Henry)

College of Veterinary Medicine, University of Missouri, USA

[email protected]

Introduction
Carriers used to solubilize taxane chemotherapy drugs cause severe hypersensitivity. Nanoparticles
can easily enter cancer cells. We recently used novel technology to formulate a nanoparticulate,
excipient-free formulation of paclitaxel (CTI52010) and determined its safety when given
intravenously to normal dogs (maximally-tolerated dose (MTD) 120 mg/m2). Our aim in this study
was to determine the pharmacokinetics, MTD, and preliminary efficacy of this drug in tumor-
bearing dogs.

Materials
Tumor-bearing dogs were recruited. Bone tumors were excluded as were dogs with significant co-
morbidities. Serial blood samples were collected after first dosing for pharmacokinetic analysis by
reverse HPLC. A modified rapid dose escalation scheme was used, increasing the dosage by 10% per
dog until a grade 2 adverse event was noted, then by 5%, and expanding cohorts when toxicity was
encountered. Tumor response was measured using RECIST criteria.

Results
Eight dogs have been enrolled, with one objective response in the urethral component of a
transitional cell carcinoma. One grade 2 neutropenia has occurred and the current dosage is 112
mg/m2 administered over 20 minutes. Pharmacokinetic data indicate a rapid initial clearing of the
drug from serum followed by an extended elimination half-life. The data for tumor-bearing animals
is similar to that for normal animals and is consistent with rapid RES clearance followed by
sustained release from a tissue reservoir.

Conclusions
This novel formulation of paclitaxel is well tolerated in dogs and no unique toxicity or
hypersensitivity has been noted. The preliminary response in one case treated below the
anticipated MTD is encouraging.
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Canine Oral Malignant Melanoma in Japan: Multi-institutional Historical Cohort Study in 165
Cases.

Tetsuya Kobayashi
(Tetsuya Kobayashi, Yuko Nakano, Ai Watabe, Tsuyoshi Kadosawa, Yusuke Ito, et al)

Japan Small Animal Cancer Center, Japan

[email protected]

Introduction
Our purpose was to evaluate the outcome and prognostic factors for canine oral malignant
melanoma (OMM) treated with surgery and/or hypofractionated radiotherapy.

Materials
Medical records from 6 institutions between 1/2000 and 12/2010 were reviewed to identify dogs
with histologically confirmed OMM. Factors evaluated for prognostic significance included age,
breed, gender, body weight, location of tumor, stage and treatment modalities. Survival was
calculated by the Kaplan-Meier method and statistical significance was set at p?0.05 using log rank
analysis for univariate analysis and Cox proportional hazard model for multivariate analysis.

Results
One-hundred sixty five dogs were identified. Median age was 12.1 years (3.0-18.0 years). Golden
Retriever was overrepresented (n=22). Median body weight was 12.2 kg (2.1-39.7kg). Overall
survival was 223 days (11-2,286 days). Median survival was 329 days (11-1,345 days) for dogs that
had surgery alone (n=51), 203 days (19-2,286 days) for hypofractionated radiotherapy alone (n=56)
and 218 days (27-1,203 days) for both surgery and hypofractionated radiotherapy (n=58). Survival
was not significantly different among the treatment groups (p= 0.3189). Significant contributors on
prognosis were age, breed, body weight, location of tumor and stage by multivariate analysis. Dogs
with OMM at the lip or the buccal mucosa had a hazard ratio of 0.5 (95%CI: 0.27-0.93) relative to
the dogs with OMM at the mandible (p=0.028).

Conclusions
These results suggest that treatment outcomes in our study are comparable to the previous reports
in the US. Treatment modalities do not appear to impact their survival.
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2 WVCC 2012 - 111 –

Continuous low-dose (metronomic) oral chemotherapy therapy of canine appendicular

osteosarcoma

J. Paul Woods
(J. Paul Woods, Sarah Boston, Anthony J Mutsaers, Dianna E Saam and Brenda Coomber)

Ontario Veterinary College University of Guelph, Canada

[email protected]

Introduction
Osteosarcoma is the most common primary bone tumour in dogs with most dogs dying of
metastatic disease despite surgery and maximum tolerated dose (MTD) adjuvant chemotherapy. In
veterinary oncology, continuous low-dose (metronomic) schedules of chemotherapy have become
more popular for patient management. Therefore, the purpose of this study was to retrospectively
investigate the effectiveness of the addition of low-dose metronomic chemotherapy following
curative-intent amputation and adjuvant carboplatinum chemotherapy to dogs with appendicular
osteosarcoma.

Materials
Dogs presented to the Ontario Veterinary College with a diagnosis of appendicular osteosarcoma
who underwent limb amputation followed by adjuvant carboplatinum chemotherapy were eligible.
Dogs completed the amputation and carboplatinum and either received no further treatment
(control); or continued with a metronomic chemotherapy protocol (treated) consisting of
cyclophosphamide (10-25 mg/m2 q 24-48h); nonsteroidal anti-inflammatory (NSAID); and
doxycycline (5 mg/kg q24h).

Results
Sixty-one dogs entered the study and 21 dogs were evaluable. The control group had 13 dogs with a
median metastasis-free interval of 178 days and a median survival time of 504 days. The treatment
group had 8 dogs with a median metastasis-free interval of 332 days (P=0.16) and a median survival
time of 340 days (P=0.55).

Conclusions
Low-dose chemotherapy had few adverse effects; however, efficacy was difficult to determine
perhaps due to lack of prospective randomization of the dogs into the groups. Future studies are
needed to determine selection of metronomic agents (timing and dosing schedules); biomarkers for
efficacy and toxicity; and combinations with standard drug dosing, newer targeted drugs, or
immunotherapy.
 n d
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SYNERGISTIC EFFECTS OF DOXORUBICIN AND DUAL INTENSITY ULTRASOUND ON TUMOR

GROWTH OF ADENOCARCINOMA BREAST CANCER IN BALB/C MICE

Amininajafi Fatemeh
(Amininajafi Fatemeh, Abbas Tavasoly, Farnosh Bagheri and Homa Soleimani)

Dept of Pathology Faculty of Veterinary Medicine University of Tehran, Iran

[email protected]

Introduction
The toxicity of the drug presents a major dose limiting factor and Doxorubicin (DOX) has
demonstrated useful effects in both early and advanced stages. The application of an
extracorporeal source of focused ultrasound (US) energy could provide a potential means of
inducing coagulative necrosis in targeted tissues without damaging .Using US exerts significant
synergistic effects on the cytotoxic action of DOX.

Materials
14 Female Balb /C mice in 4-5 weeks age were used (7 mice for treatment and control groups).
Adenocarcinoma of breast transplanted to animals of treatment group. 2 weeks later animals which
had large enough growth of tumors treated by dual US after DOX injection.

Results
This research revealed the significant effect of co-administration of ultrasound with doxorubicin on
the tumor size and also survival period in breast cancer.

Conclusions
The combined use of the drug and dual US can increase the survival period compare of method
which used it twice. Moreover the drug plus dual US is more survival period rather than other drug
plus US groups. According to the pathological observations, the area of necrotic foci in group which
used drug plus dual Us group was much more comparable to the drug with out US group.
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2 WVCC 2012 - 113 –

MicroRNA expression for classification of canine histiocytic sarcomas

Jesus Aguirre-Hernandez
(Jesus Aguirre-Hernandez, Jane, M., Tessa M Hoather, David R Sargan and Fernando Constantino-
Casas)

Dept of Veterinary Medicine, University of Cambridge, UK

[email protected]

Introduction
Flat-coated retrievers have a high incidence of soft tissue sarcomas, particularly histiocytic sarcoma
(HS). Among the latter, two forms can be recognised: subcutaneous and visceral. To aid the
diagnosis, classification and prognosis of these tumours and to elucidate the role of certain genes in
their development, we studied microRNA (miRNA) expression in formalin fixed paraffin embedded
samples (FFPE).

Materials
Eighteen FFPE samples were studied: 7 visceral HS, 6 subcutaneous HS, 4 histiocytomas and 1 high
grade soft tissue sarcoma (STS_H). For one of the visceral HS, and for the STS_H, frozen tissue was
also studied. Twenty one miRNAs were studied using quantitative real time PCR and the data was
analysed by an unsupervised classification approach and by principal component analysis.

Results
The expression profile of histiocytomas was different to the rest of the tumours. The profile of the
STS_H was also distinctive. The HS were grouped in several clusters which included both visceral
and subcutaneous tumours, except for one cluster where only visceral tumours were represented.
The miRNA expression profiles of the frozen tissues were more similar to the profiles of the
corresponding FFPE sections than to that of other samples.

Conclusions
MiRNA expression profiling correctly distinguished histiocytomas, HS and a STS_H. This technique
did not lead to the classification of HS according to their site of presentation. Instead, at least 3
different HS groups could be distinguished; which would be consistent with the recognition of
several dendritic cell lineages.
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Relative expression of fibroblast growth factor receptors in feline sarcomas

Viviana Le Donne
(Viviana Le Donne, Stacey A Snyder, Julie C Fisher and Marlene L Hauck)

University of Sassari, Italy / North Carolina State University USA

[email protected]

Introduction
Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors involved in many
physiological processes as well as cancer development and progression. At least two splice variants
(IIIb and IIIc) have been described for FGFR1-3. Expression switching of FGFR2IIIb to the IIIc isoform
relates to epithelial to mesenchymal transition in human prostate and bladder cancer and leads to
a more invasive and metastatic tumor phenotype. While extensive research investigated the role of
FGFRs in epithelial tumors, very little is known about their role and expression in sarcomas. Our
hypothesis was that feline injection site sarcomas (ISSa) and spontaneous fibrosarcomas (FSA)
would have differential expression of the FGFRs and their splice variants.

Materials
Expression levels of mRNA for these genes were evaluated by RT-PCR using cDNA from six cell lines
(four ISSa and two FSA), eight tumor samples (five ISSa and three spontaneous FSA) stored in RNA
later at -80&deg;C and forty-nine FFPE samples (39 ISSa and 10 FSA). The relative expression
software tool was used to obtain relative quantification by normalization against expression of
RPL17, YWHAZ and PPIA genes.

Results
FGFR1IIIb, FGFR1IIIc and FGFR4 were expressed in all the evaluated cell lines and tumor samples.
No statistically significant difference was found for FGFR1, FGFR2IIIb, FGFR3 and FGFR4. FGFR2IIIc
was up-regulated in ISSa if compared to FSA(P=0.028).

Conclusions
Our study demonstrated that FGFR2IIIc is differentially expressed in feline ISSa compared to FSA.
More research is needed to evaluate if different expression levels are related to the more
malignant behavior of ISSa.
 n d
2 WVCC 2012 - 115 –

Multidrug resistance membrane transporter and VEGF pattern in lymh nodes of dogs with
lymphoma and with mammary gland tumours in comparison to their normal counterparts

Peter Vajdovich
(Peter Vajdovich, Bernadett Szabó and Zsófia Koltai)

Szent István University, Faculty of Veterinary Science, Department of Internal Medicine and Clinics,
Budapest, Hungary

[email protected]

Introduction
Both ABC membarne transporters and VEGF are important factors in the resistance against
chemotherapy and the development of metastatic potential of the tumours. The aim was to
examine these in the two most frequent canine neoplasia (lymphoma, mammary carcinoma).

Materials
Dogs with lymphoma (n= 36) and others with mammary gland carcinoma (n=7) were examined. M-
RNA expression of three ABC-transporters (MDR1, mrp1, BCRP) and VEGF was quantified. Lymph
nodes of healthy dogs (n=7) and the healthy counterparts of mammary glands of the animals with
mammary gland neoplasia was examined, too.

Results
M-RNA expression in lymph nodes of healthy dogs was not different than in lymh nodes with
lymphoma in case of any of the transporters, but MDR1 was increased in relapsed compared to the
primary tumour. Dogs which showed increased sensitivity to cytostatic treatment had significantly
less MDR1, mrp1, and BCRP compared to controls. MDR1 (r=-0.62), BCRP (r= -0.55) and VEGF (-
0.72) expression was negatively correlating with the survival time, and MDR1 expression with the
relapse free period (r=-0.59). Mammary carcinomas had significantly higher MDR1, and mrp1
expression than in the normal counterparts. VEGF was significantly higher in lymphoma than in
control lymph nodes, while it was not altered in mammary tumours compared to the normal ones.

Conclusions
Tumour cells in lymphoma will develop multidrug resistance (MDR) during cytostatic treatment
whereas mammary gland neoplasia are developed with MDR. VEGF expression in lymphoma might
provides a new target for treatment.
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The study of MDM2 protein by immunohistochemistry and its promoter gene polymorphism in
canine tumors

Annahita Rezaie
(Annahita Rezaie and Mohammad Reza Tabandeh)

Pathobiology department, Faculty of Veterinary Medicine, Shahid Chamran University, Iran

[email protected]

Introduction
MDM2 is an ubiquitin ligase that suppresses the activity of P53 through ubiquitinization. The
objective of this study was to examine the MDM2 protein expression by immunohistochemistry and
genetic polymorphism in MDM2 gene by polymerase chain reaction (PCR) and direct DNA
sequencing.

Materials
In this research 20 samples of canine tumors were collected and after routin histopathology
inspection, immunohistochemistry staining and PCR were carried out.

Results
Histopathologic results revealed 15 mammary gland carcinoma, 3 lymphoma, 1 T.V.T and 1
trichoplastoma. Results of semiquantitative assessment of MDM2 onchoprotein was13 (65%)
positive from 20 samples which were including 11 mammary carcinoma, 1 lymphoma and 1
trichoblastoma. Assessment of canine MDM2 gene promoter revealed 98% homologus with human
and located on 12 chromosome. The results of PCR showed 21.05% single nucleotid polymorphism -
(19 C-A, 53 A-T, 18 G-C, 246 C-A, 292 T-G, 327 A-T).

Conclusions
These findings suggest that the MDM2 promoter polymorphism is associated with canine mammary
gland carcinoma.
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2 WVCC 2012 - 117 –

In vitro and in vivo lung carcinogenesis model induced by NNK: a model for understanding the
role of Cx 43 during the process

Gregory MENNECIER
(Gregory Mennecier, Luciana Neves Torres, Daniel Sanches, Lucas Martin Chaible, Claudia Mori et
al)

Laboratory of Oncology and pathology, Department of Pathology, School of Veterinary Medicine

and Animal Science, University of São Paulo, Brazil

[email protected]

Introduction
For long decades ago, GJIC disruption has been reported to play a role in tumoral progression in
solid tumors. A constitutive GJIC protein, the Cx43 seems to be involved during the different
hallmarks described for solid tumors progression as well cell growth, invasion and metastasis
capacities, intravasion and extravasion... However the several in vitro and in vivo studies can be
contradictory and only attribute a potentially tumor suppressor role for the Cx43.

Materials
In order to understand the implication of Cx43 during tumorigenesis, we proposed to construct an
in vitro lung carcinogenesis model. For this purpose, we decided to use tumoral alveolar epithelial
E10 cells immortalized from epithelial alveolar type II cells of BALB/c lung explants and apply the
nicotine carcinogen derivate, the NNK (100pM) during 20 cycles. Thereby, the tumorigenicity
characteristics of 4 different NNK transformed clones could be analyzed following the Cx43 state
during this process.

Results
Normal E10 cells, present a normal proliferation rate. These cells didn’t show any migration and
neoplastic characteristics in vitro. The Cx43 are expressed at the plasma membrane of E10 cells.
However, the NNK-transformed clones showed a disruption of the cell growth control, migration
capacity and a epithelila to mesenchymal transition, with a cytoplasmatic Cx43. Only the E10-
NNK20 clones showed metastasis capacity after subcutaneous inoculation into mice, where 100%
of animals showed lymph nodes and lung metastasis with cytoplasmatic Cx43.

Conclusions
This model will be use to understand the in vitro role of Cx43 for tumoral initiation, progression and
for in vivo extravasion and metastasis.
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Molecular interactions between macrophages and cancer cells growing as a co-culture in vitro

Magdalena Król
(Magdalena Król, Margorzata Gajewska, Alicja Majewska, Tomasz Motyl and Karol Marcin
Pawlowski)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences - WULS, Warsaw, Poland

[email protected]

Introduction
Solid tumors comprise various cells which regulate tumor growth and metastasis. Macrophages are
probably the most important element of the tumor microenvironment. However, the molecular
mechanism, that guides tumor environment toward a tumor-promoting phenotype, still remains
unknown.

Materials
The co-culture of cancer cells and macrophages was established. Having sorted the cells, global
gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The
results were analyzed using Gene Spring software. These results were further confirmed using real-
time qPCR and confocal microscopy.

Results
As the microarray analysis brought a large set of up/down- regulated genes to our attention, this
study focused only on the ones involved in over-manifested pathways that are
activated/inactivated whilst co-culturing cancer cells and macrophages. Among the up-regulated
genes in the cancer cell lines 13 highly over-manifested pathways were detected. The most
important pathways for us were: inflammation mediated by chemokine and cytokine, macrophages
activation, angiogenesis and apoptosis signaling. The up-regulated genes in the macrophages were
involved in only three significantly over-manifested pathways: apoptosis, integrin and angiogenesis.
The microarray analysis revealed that under co-culture (cancer cells with macrophages) the
myeloid-specific antigen expression in cancer cells as well as cytokine/chemokine genes expression
was initiated.

Conclusions
The presence of macrophages in the cancer environment induces acquisition of the macrophage
phenotype in cancer cells (specific antigens expression). We presume that cancer cells also acquire
other myeloid features, such as: capabilities of cell rolling, migration and invasion. It may be the
result of myeloid-cancer cell hybrid formation or cancer cells mimicking macrophages phenotype.
 n d
2 WVCC 2012 - 119 –

Tumor-associated carbohydrate antigen 15-3 is expressed in canine mammary cancer tissues and
cell lines and its serum level correlates with the histological grade of malignancy: the beginning of
a new perspective in veterinary oncology?

Magdalena Król
(Magdalena Król, Elisabetta Manuali, Antonio De Giuseppe, Francesco Feliziani, Katia Forti et al)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences - WULS, Warsaw, Poland

[email protected]

Introduction
Carbohydrate antigen 15-3 is aberrantly over-expressed in human mammary neoplasm and is one
of the most widely used serum tumour marker in women with breast cancer. The aims of the study
of CA 15-3 and its expression in canine mammary cancer tissues and cell lines railed us on an
investigation of the antigenic analogous of human and canine.

Materials
Immunohistochemical expression of CA 15-3 was evaluated in 7 canine mammary cancer cell lines
and 50 malignant mammary tumours. As positive controls, human breast carcinoma cell line
(MCF7) and tissue were used. Serum CA 15-3 activity was measured by means of an automatic
analyser system using a chemiluminescent method.

Results
Immunocytochemical analysis revealed CA 15-3 expression in all of the examined canine mammary
cancer cell lines. In tissues, immunohistochemical staining pattern was observed in 34 (68%) of
malignant tumours. According to the Elston and Ellis grading method, a high statistical correlation
(P=0.0019) between serum CA 15-3 levels and the degree of tumour differentiation was shown
which indicates that the values of this serum marker increase as tumour stages progress.

Conclusions
The results of this study reveal that CA 15-3 is expressed both in canine mammary tumour cell lines
and tissues and that serum levels significantly correlate with the histological grade of malignancy.
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The expression and role of ghrelin in the cancer cells isolated from canine mammary gland tumor.

Kinga Majchrzak
(Kinga Majchrzak, Katarzyna Szyszko, Joanna Mucha, Tomasz Motyl, Magdalena Król and Karol
Marcin Pawlowski)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences – WULS, Warsaw, Poland

[email protected]

Introduction
Ghrelin (GHRL) is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are
often co-expressed in multiple tumors and cancer cell lines what can indicate that the GHRL/GHS-R
axis may have an important role in the tumor growth and progression.

Materials
The aim of the studies was to assess the expression and role of ghrelin in antiapoptotic potential
and in proliferation of cancer cells. The expression of ghrelin and its receptor was assessed using
Real-time qPCR and immunohistochemistry in two adenocarcinoma cell lines isolated from canine
mammary gland (CMT-W1, CMT-W2) and two cell lines isolated from their metastases to the lungs
(CMT-W1M, CMT-W2M). For apoptosis analysis flow cytometry was used (FACS Aria II) and the
Annexin V and propidium iodide dual staining was applied. Cell proliferation was evaluated by MTT
assay and BrdU incorporation test.

Results
All examined cell lines showed the expression of ghrelin and GHS-receptor. Ghrelin treatment (1
and 10nM) significantly decreased the number of apoptotic cells whereas the administration of
antagonist of ghrelin receptor ([D-Lys3]-GHRP6) significantly increased the number of apoptotic
cells. Moreover ghrelin treatment (1, 10nM) stimulated cancer cells proliferation. The ghrelin
receptor antagonist ([D-Lys3]-GHRP6) completely abolished the stimulatory effect of ghrelin on cell
proliferation.

Conclusions
Our findings suggest that ghrelin/GHS-R signaling tilts the balance between the cellular control of
proliferation and death in canine mammary cancer cell lines and their metastases to the lungs
promoting proliferation and decreasing apoptosis.
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2 WVCC 2012 - 121 –

The anti-tumor activity of Trastuzumab in HER2-overexpressing feline mammary cancer cell lines

Fernando Ferreira
(Fernando Ferreira, Maria João Costa Soares, Margarida Pires Simões, Jorge Jesus Correia, Fernando
Costa Ferreira, Takayuki Nakagawa and Nobuo)

CIISA, Veterinary Medicine Faculty, TU Lisbon, Portugal

[email protected]

Introduction
The HER2 overexpressed oncoprotein in human breast cancer is associated with disease
progression and poor prognosis. Besides anthracycline-based therapy, a specific immunotherapy
with an anti-HER2 antibody (Trastuzumab, Herceptin®) is currently available, increasing survival
period in these cancer patients. Recent results obtained by us and others, revealed that fHER2-
overexpression is present in 30% of feline mammary carcinoma (FMC) cases, sharing a similar
incidence with women. The FMC-HER2+ high clinical frequency and the extensive HER2 protein
homology amongst human and cat, lead us to evaluate anti-tumor activity of the commercial
antibody in feline tumor cells.

Materials
FMCp, FMCm and FYCp were the feline cell lines (HER2 expression levels previously described)
submitted to several Trastuzumab concentrations (100nM, 200nM) and different time exposures
(24h, 48h and 72h). As positive control, a well-characterized HER2-overexpressing human cell line,
SKBR3, was used. In vitro antibody effects were measured by MTT assay and Trypan blue staining.
Cellular apoptotic level was analyzed through TUNEL assay (DNA fragmentation) and Western blot
(activated caspases).

Results
The HER2-overexpressing feline cell lines (FMCm, FYCp) showed pronounced decrease in their
metabolism, reduced mitotic index and augmented apoptosis, as in human positive control. The
non-overexpressing HER2 feline cell line (FMCp) was not significantly affected when submitted to
the highest antibody concentration nor under the most extended incubation period with
Herceptin®.

Conclusions
Obtained results suggest that Trastuzumab has a selective anti-tumor effect in HER2+ feline cancer
cells by recognizing a homologue extracellular region of the receptor, providing a strong possibility
for its future use in feline oncological immunotherapy.
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Review of US Cases Treated with Masitinib - Compassionate Use Phase

Barbara Kitchell
(Barbara Kitchell, Marilia Takada, Jennifer Short, ,Albert Ahn and Francois Montestruc)

Michigan State University, USA

[email protected]

Introduction
Masitinib Mesylate (Masivet in US) was licensed in the US on December 16, 2010. Between July 1,
2009 and December 16, 2010, drug was imported into the US on an FDA exemption
(‘compassionate use’) basis for individual dogs. Retrospective data was collected from dogs treated
during this period.

Materials
Data was solicited from participating hospitals based on the AB Science data base of drug
shipments. A 3 page questionaire was completed for each case. This queried histologic diagnosis,
signalment, prior therapy, drug dose, worst toxicity, response, and time to progression.

Results
A total of 545 patient doses were shipped to 203 institutions. At time of this abstract, 18
institutions provided data on 80 dogs treated for MCT. Mean dose of Masivet was 11.1 mg/kg/day.
Responses seen were 24 CR, 14 PR, and 14 SD (64% BRR)with median TTP 70 days (range 1-489
days). Reports from 96 dogs treated for non-MCT indications included: lymphoma (n=22);
osteosarcoma (n=14); carcinoma (n=14); melanoma (n=12); hemangiosarcoma (n=12); anal sac
adenocarcinoma (n=8);soft tissue sarcoma (n=7); multiple myeloma (n=4); squamous cell carcinoma
(n=3). Responses reported for 70/96 dogs included: 6 CR (3 LSA, 2 STS, 1 ACA); 9 PR (2 each
AGASACA, ACA, 1 HSA, OSA, SCC, LSA, MM);and 19 SD (5 melanoma, 4 LSA, 3 each OSA, ACA, 1 HSA,
SCC) Time to progression for non-MCT responders was 182 d for CR and 95 d for CR, PR + SD (Range
4 - 421 d).

Conclusions
Further exploration of masitinib for mast cell and non-mast cell indications is warranted.
 n d
2 WVCC 2012 - 123 –

The gene expression profiles of cancer cells induced by carcinoma-associated fibroblasts (CAFs)
grown as a co-culture in vitro

Magdalena Król
(Magdalena Król, Katarzyna Szyszko, Michal Jank, Tomasz Motyl, Karol Marcin Pawlowski et al)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences - WULS, Warsaw, Poland

[email protected]

Introduction
It is supposed that fibroblasts present in a tumour microenvironment increase cancer invasiveness
and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the
current study was designed to assess changes in gene expression in five various cancer cell lines
grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.

Materials
A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-
culture of cancer cells and the CAFs was established and maintained for 72 hrs. Having sorted the
cells, a global gene expression in cancer cells using DNA microarrays was examined. An analysis of
differential expression was performed using linear methods for microarrays (limma package in
Bioconductor software). The PANTHER binomial statistics tool was applied to determine statistically
over-manifested pathways (p<0.05). These results were further confirmed using real-time qPCR.

Results
The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the
cancer cells grown in a co-culture with the CAFs in comparison to control conditions. Bulk of the up-
regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal
transition (EMT) and generally take part in the developmental processes.

Conclusions
The results of the current study showed that the co-culturing of cancer cells and the CAFs caused
significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment
of cancer cells promotes adhesion, angiogenesis and EMT.
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STUDY OF SURGICAL TREATMENT OF ORAL MALIGNANT MELANOMA AND THERAPEUTIC

ANTICANCER POOL VACCINE IN DOGS

Claudia Ronca Felizzola

(Claudia Ronca Felizzola and Suzana Orsinni Machado de Souza)

Clinica Cirurgica Oncologica, University of Sao Paulo, Brazil

[email protected]

Introduction
Oral malignant melanoma (OMM) is most frequent cancer in the mouth of the dogs, with great
metastatic disease, and resistant chemotherapy. Dogs present spontaneous oral melanoma and
could be a model for cancer immunotherapy.The aim of this study was to evaluate a radical surgical
treatment plus pool vaccine of dogs with diagnosis of oral malignant melanoma, estimate
recurrence, metastasis quality of life and survival time.

Materials
Eighteen dogs from several breeds and mongrel, male and female, with ages raging from 7 a 15
years, present oral malignant melanoma were used for this present study. TMN classification is one
dog stage II, 10 dogs stage III, and seven dogs stage IV. Dogs were examined at many Private
Surgical Service of São Paulo (Brazil), for diagnosis and treatment of oral lesions (OMM). All dogs
had radical surgery and received pool vaccine (three doses or more).

Results
Two dogs had recurrence, and two pulmonary metastasis (these dogs were euthanized). The
average of survival time until present moment is 483 days. In the first year, more than 70% of dogs
live with quality life.

Conclusions
Furthermore, this study provides new cancer therapy vaccine, for use in treatment of human and
animal cancer, and could be use the dog as model for treatment malignant melanoma in men
 n d
2 WVCC 2012 - 125 –

The efficacy and toxicity of Dexamethasone, Melphalan, Actinomycin D, Cytosine Arabinoside

(DMAC) protocol for relatively small breed dogs.

Fukiko Oshima
(Fukiko Oshima, Tetsuya Kobayashi, Eri Fukazawa, Yuko Nakano, Takashi Higuchi et al)

Japan Small Animal Cancer Center, Yumiko Kagawa,North Labo, Japan

[email protected]

Introduction
The dexamethasone, melphalan, actinomycin-D, cytosine arabinoside protocol (DMAC) was
reported to be effective for canine refractory lymphoma. Our purpose was to evaluate the efficacy
and toxicity of DMAC in dogs with lymphoma that had relapsed.

Materials
Medical records from the Japan Small Animal Cancer Center between 7/2006 to 4/2011 were
reviewed to identify dogs with multicentric lymphoma treated with the UW-25 protocol followed
by DMAC. Hematologic and gastrointestinal adverse events were evaluated according to VCOG-
CTCAE (v1.0) criteria.

Results
Sixteen dogs were identified. All were pure breed. Median body weight was 14.1 kg (4.5-39.2kg).
Ten of sixteen (62.5%) had lymphoma of B-cell origin and the other 6 (37.5%) had T-cell lymphoma.
Overall response rate was 43.7% (CR=31.2%, PR=12.5%) and median response duration was 21days
(14-808). One dog died due to tumor progression 2 days after initiation of DMAC and 15 dogs were
evaluated for adverse events. Grade I or higher neutropenia, thrombocytopenia, vomiting and
diarrhea were observed in 10/15 (66.6%), 12/15 (80.0%), 4/15 (26.6%) and 8/15 (53.3%) dogs,
respectively. Sepsis was seen in 6/15 (40.0%) at a median of 9 days after initiation of DMAC.
Treatment delay on day-7 was necessary due to adverse events in 6/11 dogs (54%).

Conclusions
Although the response rate of DMAC is comparable to other rescue protocols, adverse events were
too high and unacceptable. Modification of the dose intensity of original DMAC might be necessary
for relatively small breed dogs.
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The expression of genes from ABC superfamily in canine mammary cancer cell lines

Katarzyna Szyszko
(Katarzyna Szyszko, Kinga Majchrzak, Joanna Mucha, Tomasz Motyl, Magdalena Król and Karol
Pawlowski)

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life

Sciences-WULS, Warsaw, Poland

[email protected]

Introduction
Chemoresistance is a main problem in human and veterinary oncology. The most important
mechanism of chemoresistance is the expression of efflux pumps from ABC superfamily. The major
efflux pumps are: P-glycoprotein (PGP), Multidrug resistance protein 1 (MRP1), Multidrug
resistance protein 3 (MRP3), Breast cancer resistance protein (BCRP).

Materials
The aim of the study was to assess the expression of efflux pumps and apoptosis in seven canine
mammary cancer cell lines in control conditionals and after the vinblastine and paclitaxel treatment
at IC50 doses. The IC50 doses of drugs were determined using MTT assay. For apoptosis analysis
flow cytometry was used and the Annexin V and propidium iodide (PI) dual staining was applied.
The expression of efflux pumps was assessed using Real Time qPCR method.

Results
All examined cell lines showed the expression of MRP1 and BCRP in control conditions. Six cell lines
showed expression of MRP3 and three of them showed the expression of PGP in control conditions.
In almost every case the expression increased significantly after the drugs treatment or appeared in
these cell lines in which it was not observed in control conditions. The number of apoptotic cells
increased after the treatment with cytostatic drugs in all examined cancer cell lines.

Conclusions
There is a wide variation of efflux pumps expression both between types of pumps, as well as
examined cancer cell lines. Moreover, the expression of efflux pumps in control conditions doesn`t
reflect the level of multidrug resistance.
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2 WVCC 2012 - 127 –

PET/CT and SPECT/CT imaging in oncological animal patients – feasibility and radiotoxicological
aspects, clinical experiences.

Lajos Balogh
(Lajos Balogh, Zita Postenyi, Veronika Haasz, Andras Polyak, Julianna Thuroczy, et al)

NRIRR, Budapest, Hungary

[email protected]

Introduction
Despite that the clinical usefulness of hybrid imaging techniques in human oncology is well
understood much less data are available from the veterinary side. We goaled to work-out PET/CT
and SPECT/CT imaging techniques for localizing, staging and follow-up veterinary oncological
patients.

Materials
Client owned dogs and cats altogether over 120 suspected or known oncological cases were
referred for 18FDG PET/CT (Phillips Brillance)and 99mTc-pertechnetate, 99mTc-MDP, 99mTc-MIBI
SPECT/CT (AnyScan, Mediso) whole body examinations. The goal was to detect primary tumour
localizations, extensions and to visualize regional and far metastases (WHO staged diagnosis. We
also collected blood samples from patients to check the possible (radio)toxicologiocal effects.

Results
18FDG PET/CT revealed very sensitive in case of a wide variety soft tissue and bone tumours eg.:
osteosarcomas, mast cell tumours, lymphomas, soft tissue sarcomas, melanomas and different
epithelial tumours. Some benign, low metabolic rate tumours (adenomas, lipomas, fibromas,
intracranial) showed much lower 18FDG-uptake where the method seems to be questionable. Few
degenerative-, inflamed- and hypertrophic tissues (arthrosis, lymph adenitis, muscle hypertrophy)
revealed intense focal uptake that could be confusing in differential diagnoses. SPECT/CT technique
found to be extremelly usefull in thyroid-, bone tumor detections and in vivo MDR examinations.

Conclusions
Whole body hybrid imaging is well-tolerated even in late stage oncological patients and provides
important information to veterinary clinicians in disease staging, monitoring response to treatment,
planning and choosing appropriate therapies, detecting recurrence and predicting prognosis.

Scientific work was supported by several national (OTKA-68376, JEDIONKO, KMOP-1.1.1-08/1-2008-

0017, GOP-1.1.1.-09/1-2010-0107) and international (IAEA-CRPs, EMIL NoE) projects.
 n d
- 128 - 2 WVCC 2012

ABSTRACTS FOR PUBLICATION ONLY

 n d
2 WVCC 2012 - 129 –

IMAGING FOLLOW-UP OF CANINE INTRACRANIAL NEOPLASIA TREATED WITH RADIATION

THERAPY

Federica Rossi
(Federica Rossi, Paola Laganga, Vito Ferdinando, Leone Simona, Cancedda Massimo Vignoli, et al)

Centro Oncologico Veterinario, Sasso Marconi (Bologna), Italy

[email protected]

Introduction
Intracranial neoplasia is one indication for radiation therapy (RT). High total doses divided in small
fractions are mandatory to reach maximal tumour control minimizing side effects. Clinical
improvement depends on early decrease of peritumoral inflammation and later tumour volume
(TV) reduction. Contrast Enhanced Computed Tomography (CECT) or Magnetic Resonance (MR) are
necessary for volume definition prior to RT and can be used to monitor TV in treated patients.

Materials
Ten dogs with intracranial neoplasia and treated with high-dose RT protocol (mean total dose of >
48 Gy divided in 20 fractions) were monitored clinically and by follow up CECT or MR. Pre-and post-
treatment TV was contoured by a radiologist on the CT, pre-treatment values were compared to
the GTV drawn by the radiation oncologist on the TPS.

Results
Treated tumours included presumed meningioma (n=5), pituitary macroadenoma (n=3) and glioma
(n=2). Dogs showed rapid clinical improvement during RT. CECT (n=9) or MRI (n=1) were performed
between 6 and 8 (n=9) or 15 months (n=1) after RT. Pre-treatment mean TV was 2,5 cm3 (range
0,87-5,8) mean GTV was 3,31 cm3. Post-treatment mean TV was 1,2 cm3 (range 0,34-2,71). A mean
volume reduction of 58% was calculated Interobserver volume definition showed a difference of
25% between radiologist and radiation oncologist. No indication of brain necrosis was observed.

Conclusions
RT was well tolerated and leaded to rapid clinical improvement and evident TV reduction. CECT and
MR allowed to objectively monitor TV before and after treatment. We suggest repeating scans
between 6 and 8 months after RT.
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MULTIMODAL THERAPEUTIC APPROACH AND INTERDISCIPLINARY CHALLENGE FOR THE

TREATMENT OF UNRESECTABLE HEAD AND NECK SQUAMOUS CELL CARCINOMA IN 6 CATS: A
PILOT STUDY

Laura Marconato
(Laura Marconato, Julia Buchholz, Paola Valenti, Barbara Kaser-Hotz, Marcel Keller and Giuliano
Bettini)

Animal Oncology and Imaging Center, Hunenberg, Switzerland

[email protected]

Introduction
Feline head and neck squamous cell carcinoma (HN-SCC) harbors a poor prognosis. The complex
anatomic location precludes aggressive surgery. Chemotherapy and radiation therapy (RT) have
been disappointing. Aim of this trial was to achieve prolonged survival while having a good quality
of life using a multimodal strategy, including medical treatment (MT), RT and surgery.

Materials
Cats with histologically confirmed, unresectable, HN-SCC with exclusion of the mandible and
without distant metastases were enrolled. After complete staging, cats underwent MT (thalidomide
2 mg/kg SID; piroxicam 0.3 mg/kg EOD and bleomycin 10 UI/m2 weekly), RT (BID fractions over 5
consecutive days; total dose 48 Gy) and surgery.

Results
Six cats were enrolled. Tumor locations were tongue (n=3), larynx (n=2) and maxilla (n=1). All cats
underwent neoadjuvant MT. Surgery was undertaken before RT in 3 cats downstaged to resectable
disease. Two cats with sublingual SCC still considered not surgically excisable after MT were
irradiated first, followed by surgery. One cat with maxillary SCC was irradiated first but developed a
primary lung carcinoma thereafter. Three cats with sublingual SCC were alive and in complete
remission after 759, 458 and 362 days. One cat with laryngeal SCC died of renal lymphoma after 51
days; one cat with maxillary SCC died of a primary lung tumor 82 days after diagnosis. In both cats
the SCC was in complete remission. Only one cat developed metastases after 144 days.

Conclusions
These preliminary results are encouraging and the possible benefit of this strategy merits further
evaluation.
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Correlation between Cytology and Histopathology in Evaluation of Canine Growths

Yogita Pawar
(Yogita Pawar, Gajanan S Khandekar and Renuka S Nehete)

Radiation Medicine Center BARC Parel Mumbai, Mumbai, India

[email protected]

Introduction
Cytology has recently been employed in diagnosis of growths in dogs and cats. However, very few
studies have addressed the accuracy of cytology in evaluation of growths in clinical setting. A
prospective study was undertaken to compare the accuracy of cytology in diagnosis of canine
growths.

Materials
Forty six dogs clinically affected with growths were taken for study. Tissue samples were collected
from each case by surgical excision for histopathology. However, for cytological evaluation, samples
were taken by fine needle aspiration, by impression smears or by scraping. The cytological smears
were fixed with ethanol, methanol or air dried and stained with Leishman stain, Wright’s stain and
Papanicolus stain. Cytological findings were correlated with gross morphology, location of tumor,
morphometric findings and histopathology. Concordance and accuracy of the cytology for diagnosis
of growths was determined using histopathology as gold standard.

Results
The gross appearance and location of tumor was helpful in diagnosis of certain tumors like cystic
and mixed type of adenocarcinoma of mammary gland. Furthermore, morphometry helped in
deciding the malignancy of tumors. Histopathological diagnosis categorized 91.30% growths as
neoplastic and 8.69% as inflammatory growths. Neoplastic growths were further classified as
malignant (69.04%) and benign (30.96%). Diagnosis obtained by cytology accurately reflected the
diagnosis obtained on histopathological examination in 100% round cell tumors, 87.7% of epithelial
cell tumors, 86.6% of mammary gland tumor, 60% of fibrosarcoma and 25% of hemangioma cases.

Conclusions
Cytological diagnosis was in agreement with histopathology which confirmed cytology as a reliable
and useful method in evaluation of canine growths.
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EARLY CLINICAL IMPRESSIONS REGARDING THE USE OF MASITINIB IN NON-MAST CELL

NEOPLASMS IN THE CANINE.

Frederick H. Drazner
(Frederick H Drazner)

Animal Specialty Services of Cook County, Des Plaines, Illinois, United States

[email protected]

Introduction
In-vitro studies utilizing cell lines of common canine neoplasms (HSA, OSA, T-Cell LSA, carcinomas,
etc.,)demonstrated increased cell death when masitinib was added to various traditional cytotoxic
chemotherapy agents (doxorubricin, vinblastine, gemcitabine and carboplatin). These studies
suggested that masitinib may be a chemosensitizer.

Materials
Masitnib and a cytotoxic agent were used as follows: 1) Splenic HSA-doxorubricin, 2) OSA-
carboplatin, 3) T-Cell LSA, 4) TCC of the urinary bladder. Dogs were treated with masitinib and a
cytotxic agent for 12 weeks and were re-evaluated at 2 week intervals and checked for toxicity.

Results
Toxicity attributed to masitinib was minimal and relatively infrequent. Dogs with Splenic HSA did
poorly and did not demonstrate any improved length of survival over doxorubricin monotherapy.
Dogs with OSA and T-Cell LSA demonstrated minimal to moderate improvement of length of
survival as compared to monotherapy with carboplatin and doxorubicin, respectively. TCC of the
urinary bladder/urethra demonstrated the most encouraging results with 2 dogs surviving 13 and
15 months,respectively and one dog with no demonstrable disease still alive at 21 months.

Conclusions
The TKI masitinib when combined with traditional cytotoxic agents was well-tolerated and
demonstrated minimal positive results for OSA and T-Cell LSA and no improvement for Splenic HAS.
The most encouraging results were seen with TCC of the urinary bladder and urethra. It is
emphasized that the case numbers were quite small in this pilot study but these preliminary results
warrant further study.
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Personalized Medicine in Veterinary Oncology

Marlene L. Hauck
(Marlene L. Hauck and Shane C Burgess)

North Carolina State University, Raleigh, United States

[email protected]

Introduction
Significant advances in cancer treatment have been through the identifying and targeting molecular
abnormalities. The last decade has seen the development of cost-effective sequencing technology
and accompanying bioinformatics tools empowering concurrent sequencing and measurement of
relative gene expression levels of all transcribed regions from individual tumors. Our hypothesis
was that individualized, specific treatment approaches could be discovered for each tumor.

Materials
The Illumina Genome Analyzer IIx was used to perform all exonic deep sequencing experiments.
Relative expression levels of all genes (expressed as “reads per kilobase of exon model per million
mapped reads” (RPKM) values), and aligned all sequences to the Canis familiaris genome utilizing
the BowTie software. These mRNAs were then annotated and relative expression levels of all mRNA
sequences analysed with Ingenuity Pathway Analysis (IPA) software. Two canine tumors were
sequenced—one osteosarcoma (OSA) and one histiocytic sarcoma. Relative expression levels for all
canine genes were determined and analyzed for differential pathway activation. In the histiocytic
sarcoma (a tumor of dendritic cell origin), we looked specifically at pathways known to be
expressed in similar cells. With the OSA there were no known expressed pathways for bone or
mesenchymal tumors; therefore the comparison was made to all cancer pathways.

Results
Several pathways were significantly overexpressed in the histiocytic sarcoma and contained
targetable nodes. All of the most highly expressed pathways in the OSA are potentially targetable.

Conclusions
These patients demonstrate the potential of an individualized approach to cancer treatment in
veterinary oncology.
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Photodynamic-Hyperthermal Chemo-Therapy with Indocyanine Green against Canine Soft Tissue

Sarcomas in Limb : 5 cases

Yoshiharu Okamoto
(Yoshiharu Okamoto, Takeshi Tsuka, Tomohiro Imagawa, Tomohiro Osaki, Masahiko Sugiyama, et
al)

Tottori University, Tottori-shi, Japan

[email protected]

Introduction
Indocyanine green (ICG) induces heat in response to light at a wavelength of 808 nm and oxygen
radical in response to light at a wavelength of 600-800 nm. A novel cancer therapy was developed
using these properties of ICG and a broad band light source instead of a diode laser, to create a
combination of photodynamic therapy and hyperthermia. This therapy was called Photodynamic
Hyperthermia (PHT). Furthermore, this double therapy was combined with local chemotherapy to
create a triple therapy, called Photodynamic Hyperthermal Chemotherapy (PHCT).

Materials
Five dogs with soft tissue sarcomas in limb were treated by PHCT. ICG was dissolved in 9 ml of
saline adjusted to pH 5.0, containing 1 ml of bleomycin or cisplatin. A broad band light source,
which emits a wavelength spectrum from 600-1,600 nm and 5,000 mW in maximum output power.
The tumor was resected and the ICG solution was injected into the resected area including the
surgical margin (2 cm). Irradiation was administered at 10 cm away from the resected area
(irradiation area: 113 cm2, 40 mW/cm2) for 20 min per 113 cm2 (53 J/cm2) just after the injection
of ICG solution. PHCT was performed over an interval of 1-2 week and repeated several times.

Results
One case had amputation because of early recurrence but four cases were saved their limbs and
recurrence was not recognized over 1 year in 3 cases out of 4 cases.

Conclusions
PHCT with ICG has a potency of useful treatment against tumor.
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Immunohistochemical study of expression of urokinase plasminogen activator (uPA) in canine

malignant mammary tumors

Annahita Rezaie
(Hannaneh Golshahi, Abbas Tavasoli and Annahita Rezaie)

Pathobiology department, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz,

Islamic Republic of Iran

[email protected]

Introduction
uPA as a member of the serine protease family has a key regulator of biologic processes implicated
in tumor cell migration, invasion, and metastasis . Determination of uPA levels has also proven to
be a useful negative prognostic marker of disease progression such as poorly human breast cancer.

Materials
uPA immunohistochemical expression was studied in 37canine malignant mammary tumor to
investigate the relationship of histopathological type, histopathological grade with the expression
of uPA.

Results
In 29 (78.4%) cases, the expression of uPA with neoplastic cell was < 10% and in 34 samples (91.9%)
uPA expression with stromal cell was < 10%. uPA expresssd in epithelial, myoepithelial cells of
carcinomas and also in mesenchymal population of carcinasarcoma, chondrosarcoma and
carcinoma arising in benign samples. Reactive fibroblast and macrophages from stromal compound
also demonstrated uPAexpression. The intensity of expression of uPA with stromal cell was
associated with histological grade (p<0.05) .but no significant relationship was detected between
uPA expression of neoplastic cell (epithelial, myoepithelial and mesenchymal cell)with histological
grade(p<0.05).

Conclusions
Increased expression of uPA in tumor stroma is associated with poor prognostic factors. Stromal
expression of uPA may a prognostic indicator for canine mammary tumor. On the basis of these
results, uPA could be considered a useful prognostic factor in dogs with mammary tumors.
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Immunohistochemical study of expression of Caveolin-1 (Cav-1) in canine malignant mammary

tumors

Annahita Rezaie
(Annahita Rezaie, Hannaneh Golshahi and Abbas Tavasoli)

Pathobiology department, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz,

Islamic Republic of Iran

[email protected]

Introduction
Caveolin-1 is thought to have an important impact on both signal transduction and mediation of
intracellular processes. It has been suggested that Caveolin-1 may contribute to certain steps of
carcinogenesis in various types of cancer.

Materials
Cav-1 immunohistochemical expression was studied in 37canine malignant mammary tumor to
investigate the relationship of histopathological type, histopathologic grade with the expression of
Cav-1.

Results
Cav-1 was expressed by epithelial cells in 33of 37 (89.2%) malignant mammary tumors. There was a
significant relationship between epithelial expression of Cav-1and histological grade of tumors
(P<0.05). No significant relationship was observed between expression of Cav-1 in myoepithelial
and mesenchymal cell with histological grade (P<0.05).

Conclusions
This study showed that Cav-1 can use as a prognostic factor and its expression is associated with
more malignant canine mammary tumors .
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Efficacy of local chemotherapy in dogs with nasal tumors

Eszter Szendi
(Eszter Szendi, Peter Vajdovich and Barbara Szalay)

Veterinary Hematology and Oncology Center, Budapest, Hungary

[email protected]

Introduction
93 dogs with intranasal tumors of various histopathologic types (carcinoma: n=26, sarcoma: n=17)
was reweived. Among these 24 had rhinotomy and 14 had local chemoterapy.

Materials
Local chemoterapy was made through the frontal sinus and nasopharyngs retrograde, and through
the nasal cavity anterograde route.The marker parameters of the progressive disease based on 3
points WHO staging system, the average point of the patients who recieved chemoterapy was 2.3,
of the operated patients was 1.81, and the untreated patients’ average was 1.87.

Results
The average survival time of all patients was 214 days. The median survival time of the patients
who got local chemoterapy was 122 days, and the surgical cases had 198 days. However, the
chemotherapy patients were significantly (p< 0.05) belonged to higher WHO-stage (average: 2.46 ±
0.66) at the first examination of the disease, than the operated patients (average: 1.81 ± 0.75). In
case of the 3.stage patients was significant (p<0.01) difference. The operated patients’ median
survival time is 35.5 and the chemotherapy patients’ 122 days.

Conclusions
Based on these examinations, all of the dogs who were treated by chemoterapy improved, and
tolerated the treatment very well. In case of the operated dogs, 21 from 24 patients improved, and
tolerated the treatment meanly. Some patients were not treated with surgery or chemoterapy,
they got only symptomatic treatment, and 2 of them showed improvement. Local chemoterapy
seems to be a chance to treat patients with higher stages. This treatment is probably more
successfull with the combination of surgery.
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STUDY OF LIVER LOBECTOMY FOR TREATMENT HEPATIC TUMORS IN DOGS.

Claudia Ronca Felizzola

(Claudia Ronca Felizzola, Silvana Pandolfi Jatene, Lydia Storte Borragina, Claudia Ferreira,and Alex
Jader)

VCS- Clinica Cirurgica Oncologica, São Paulo-SP, Brazil

[email protected]

Introduction
Hepatic neoplasm is an uncommon tumor in domestic animals. In dogs, the frequency is 2% of all
neoplasm. Hepatocellular carcinoma (HCC) is the most common primary hepatic tumor in dogs. The
treatment of choice is hepatic lobectomy, and the prognosis is good. The aim of this study was
clinic pathologic and follow up features of canine primary hepatic neoplasm.

Materials
This study was realized on Oncological Private Clinic in São Paulo- Brazil, between 2007 until 2011.
All 13 dogs diagnosed hepatic neoplasia; there were analyzed clinical, imaging, laboratories tests,
surgical treatment, histological types of tumor and follow up.

Results
Thirteen dogs had hepatic tumor (five males, eight females), histological are: hepatocellular
carcinoma HCC (n=9), hemangiosarcoma HSA (n=2), hepatic sarcoma HS (n=1), hepatic adenoma
(n=1). All dogs have high alanine aminotransferase and aspartate aminotransferase activities. The
median survival time was X= 449 days (2 days until 1460 days), left medial lobo is a local more
common. Before discovery hepatic neoplasia, two dogs have a malignant melanoma and three dogs
have mammary carcinoma, after a lobectomy one dog has transitional cell carcinoma (TCC) and
small intestinal carcinoma. Metastasis only one dog has HSA, and three cases died because heart
failure.

Conclusions
Dogs with hepatic neoplasia are recommended a liver lobectomy. The tumor control excellent, no
local recurrence and low metastasis.
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PALLIATIVE RADIATION THERAPY AND FOLLOW UP LOMUSTINE FOR TREAMTENT OF HISTIOCYIC

SARCOMAS IN DOGS

Janean Fidel
(Janean Fidel, Chrisite Anderson, Tamara Wills, Gary Haldorson and Melanie Spoor)

Washington State University, Pullman, United States

[email protected]

Introduction
Published survival times for localized histioctyic sarcomas have been 7-9 months and often involve
aggressive surgery including amputation. Radiation therapy can also be used to treat histiocytic
tumors. This study was undertaken to document the effectiveness of a combination of palliative
radiation and lomustine in dogs with histiocytic sarcoma.

Materials
Staging consisted of CBC, chemistry panel, thoracic radiographs, abdominal ultrasound and FNA of
effected lymph nodes. Radiation using 5 x 6Gy or 4 x 6Gy was given followed by Lomustine at 60
mg/m2 every 4 weeks for four doses with possible continuation.

Results
23 dogs with histiocytic sarcoma in a location amenable to radiation therapy were treated. Mean
age was 8.1 years; dogs included 14 FS, 8 MC, and 1 MI; breeds were retrievers (13), Rottweilers
(3), Australian shepherds (2), Bernese mountain dogs (2), other pure bred dogs (3) and 1 mixed.
Stages included I (n=9), II (n=8) and III (n=6). Median overall survival time was a 295 days (26-1174
days). Significant predictors of survival were stage (P=0.0003) with median survival of stage I, II and
III being 615, 297, and 144 days respectively. Flat coated retrievers and golden retrievers had
significantly shorter median survivals than others (169, 183, and 325 days respectively, P= 0.0239).
Dogs that completed Lomustine lived significantly longer (325 days versus 72 days, P=0.007) as did
dogs that received additional doses of Lomustine (364 days versus 163 days, P=0.0014).

Conclusions
Radiation of localized histiocytic sarcomas plus Lomustine gives long survivals and likely as long as
therapies involving surgical removal.
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Influence of clinical stage at the survival time of dogs with malignant mammary tumours treated
by surgery and adjuvant chemotherapy.

Sobral, Renata A.
(Sobral Renata A, Rodrigo Ubukata, Suzana Honda Battaglia, Juliana Cirillo, Fernanda Duarte dos
Santos Malatesta, et al)

PROVET - Instituto de Diagnósticos e Especialidades Veterinárias, São Paulo, Brazil

[email protected]

Introduction
Malignant mammary tumours are common tumours in old, not spayed, female dogs. This study
presents the influence of clinical stage at the survival time of patients treated by surgery and by
surgery and chemotherapy during a follow up minimum of 24 months.

Materials
48 female dogs were attended at Instituto de Diagnósticos e Especialidades Veterinárias (PROVET)
and, at Hospital Veterinário da Universidade Paulista de Ciências Agrárias; São Paulo, Brazil.
Patients were evaluated by physical examination and screening for metastasis. Surgical was
performed and, chemotherapy started after surgery. Cyclophosphamide plus flurouracil was given
weekly/4-6 times and, doxorubicin plus cyclophosphamide was given each 3 weeks/3 to 6 times.

Results
The middle age of the patients was 10 years old. Clinical stage were classified as, ECI (22,9%); ECI/II
(43,7%); ECIII (22,9%) and ECIV (10%). Thirty one patients (64,5%) died at a middle follow up of 15
months, almost 50% of them were EC III or ECIV. Alive patients had histologic grade tumours
classified as, grade1 (68,7%) and grade2 (31,2%) while, 37,5% of died patients were grade 3. Most
common types tumours were tubular (16/4), complex (7/48) and solid (6/48). Forty six patients
were treated by surgery and, from them, 47,9% (23/48) received chemotherapy. Middle survival
time for patients which received cyclophosphamide and doxorubicin plus cyclophosphamide was,
respectively, 42 and 19 months. The middle survival time of patients which did not receive
chemotherapy was 29 months.

Conclusions
Adjuvant chemotherapy seems increased the survival of patients but, not for patients with
metastatic disease (ECIV).
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Evaluation of Cox-2 expression in 147 canine malignant tumours of distinct histological types

Felisbina Queiroga
(Felisbina Queiroga, Felisbina,Luisa,Queiroga, Teresa Raposo, Justina Prada, Isabel Pires and Hugo
Gregório)

University of Trás-os-Montes and Alto Douro, Vila Real, Portugal

[email protected]

Introduction
Cyclooxygenase-2 (Cox-2) expression in canine tumours has been subject of intense investigation
for the last decade. The present report aims to illustrate Cox-2 expression in a series of malignant
tumours that include several histological types not described so far.

Materials
147 Malignant tumours were included, grouped according to their anatomic location. Cox-2
monoclonal antibody (Labvision, clone SP21) was used for immunohistochemistry. Tumours were
classified as positive when more than 10% of the cells showed Cox-2 immunolabelling.

Results
We found 117 Cox-2 positive cases (79,6%). Alimentary tract tumours (n=17) were all positive
(gastric and liver carcinomas, mesotelioma, oral SCC, melanomas and mastocytomas ). Bone and
joint tumours 4/5 cases were positive. Tyroid carcinomas (n=4) demonstrated positivity in 3 cases.
Genital tumours (n=13) showed positivity in 10 cases (TVT, Sertoli, seminoma, Leydig, and ovaric
tumours and vulvar mastocytoma). In hematopoietic tumours (n=7), lymphomas were negative
(n=4) and 1/3 splenic hemangiossarcomas positive. In mammary tumours 42/49 cases were
positive. In nervous system (n=2), 1 meningioma was positive and 1 oligodendroglioma negative. In
ocular tumours (n=5), 1 iridociliar carcinoma and 2 melanomas were positive. Respiratory system
tumours (n=4) were positive (2 nasal and 2 pulmonary carcinomas). Among cutaneous and soft
tissue tumours (n=40), 31 were positive: [perianal carcinomas (1/2); mast cell tumours (14/15);
melanomas (7/8), soft tissue sarcomas (7/11) and 2 SCC]. The only renal adenocarcinoma analyzed
was positive for Cox-2.

Conclusions
Cox-2 is widely expressed in canine malignant tumours, strengthening the idea that specific Cox-2
inhibitors might be of usefulness in the treatment of these neoplasms.
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Canine neoplasms of the spleen: retrospective analysis of 23 surgical cases.

Luciana Oliveira de Oliveira

(Luciana Oliveira de Oliveira, Luciane Cristina Vieira, Kelly Cristine Rocha da Silva Ferreira, Suzana
Vargas Cheuiche, Daniel Guimarães Gerardi and Daiana Rauber)

Veterinary Hospital, Federal University of Rio Grande do Sul, Porto Alegre, Brazil

[email protected]

Introduction
The purposes of this study were to document clinical signs, histopathological data and survival
times from dogs submitted to splenectomies due to splenic masses.

Materials
The cases in this study were selected from a review of surgical records of splenectomies in dogs.
Data on breed, sex, age, clinical signs, clinical stage, histopathologic diagnosis and survival time
were obtained for 23 dogs.

Results
Twenty-three cases of splenic tumors were analyzed. Most dogs were mixed breed (26.09%),
females (82.61%), and median age 9.7 years (6-17 years). Age at diagnosis for malignant tumors
was 9 years, for benign tumors it was 12.2 years. Common clinical signs were lethargy, abdominal
distention and pain. Four dogs had splenic nodules detected in check-up ultrasonographies. From
the 23 cases, 18 were malignant (11 hemangiosarcomas, 4 indifferentiated sarcomas, 2 lymphomas
and 1 fibrosarcoma) and 5 were benign (2 lymphoid hyperplasias, 2 hemangiomas and 1 thrombotic
nodule). Most malignant tumors were clinical stage T2 (43.75%). Four dogs with splenic malignant
masses died in the immediate postoperative period (30 days), 2 dogs died within 6 months and 4
dogs within 12 months. At the cut point of the study, three dogs were still alive at 6 months
postoperatively, 2 dogs at 12 months, 1 at 2 years. Only 2 dogs survived more than 2 years.

Conclusions
Results of this retrospective review indicate that most canine splenic neoplasms are malignant,
hemangiosarcoma accounts for the majority of splenic masses and it has a poor prognosis in most
cases.
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Imatinib Mesylate as Treatment on Mast Cell Tumors (MCT) in Dogs - Preliminary Study

Thaís Rodrigues Macedo

(Thaís Rodrigues Macedo, Samanta Rios Melo, Thais Andrade Costa-Casagrande, Julia Maria
Matera, Maria Lucia Zaidan Dagli and Maria Luiza Franchini)

Department of Surgery - School of Veterinary Medicine and Animal Science – University of São
Paulo, Brazil, São Paulo, Brazil

[email protected]

Introduction
The MCT is the most frequently canine cutaneous malignancy, with a inpredict biological behaviour.
Mutation on exon 11 of proto-oncogene c-kit, which induces constitutive phosphorylation of KIT
have been considered one of the mechanisms for the development or progression of mast cell
tumor in dogs. Imatinib Mesylate is a small-molecule tyrosine kinase inhibitor that selectively
inhibits proliferation and induces apoptosis in Bcr-Abl positive cell. The goal of this study is to
evaluate the therapeuthic potential of imatinib mesylate in canine MCT.

Materials
Six dogs with MCT grade II or III, with single or multiple tumors and not resecteable were treated
with imatinib mesylate, at an oral dose of 10mg/kg daily for 60 days. They were evaluated for KIT
expression. Responses of MCT to imatinib were assessed based on the RECIST guidelines. We used
as criteria the VCOG-CTCAE to evaluate toxicity of chemotherapy security.

Results
One (16.7%) dog had completely remission, four (66.7%) dogs showed a stable disease and one
(16.7%) progression of the disease. Only one (16.7%) dog had his treatment discontinued, he
showed vomitus grade III, as side-effects were observed neutropenia grade I (16.7%) and anorexia
grade I (16.7%). Five animals were classified as KIT II and one animal as KIT III.

Conclusions
Our data suggest that imatinib mesylate has activity against the MCT, but we plan to enroll
additional dogs to confirm our results.
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Body Condition Score in cats with feline injection-site sarcoma (FISS).

Carolina Scarpa Carneiro

(Carolina Scarpa Carneiro, Valdecir Marvulle, Maria Lucia Zaidan Dagli and Julia Maria Matera)

Department of Surgery - School of Veterinary Medicine and Animal Science - University of São Paulo
(SVMAS /USP), Brazil, São Paulo, Brazil

[email protected]

Introduction
The FISS is an aggressive neoplasia, with no efficient treatment. The major of feline patients are
neutered. The persistent inflammation is related with FISS pathogenesis. The overweight may be
related to the presence of FISS.

Materials
The body condition score (BCS) was performed and correlated in two group: FISS patients and feline
population of the Veterinary Hospital – SVMAS/USP – Brazil. 20 FISS patients were analyzed. Was
used the table of BCS, validated by Laflamme (1997), ranges from 1 to 9: -1-4 being
undernourished; 5 ideal weight; 6-9 overnutrition. The BCS was suggested by the researcher; the
weight and age were recorded. All animals were neutered. The data analyzed were BCS, weight and
age. The data presents normal distribution by the test Shapiro-Wilk, and underwent to t-Student
test.

Results
The age average was 9 years (standard derivation-SD= 3.3) in FISS patients and 10 years (SD=3.8)
with p=0.28. Weight average was higher in FISS group (5.6kg, SD=1) than in control group (3.7kg,
SD=1), with p<0.001. The BCS average in FISS group was 6.7 (SD=1.6), whereas in control group
these average was 4.8 (SD=1.8), with p=0.001.

Conclusions
Weight and BCS were greater in FISS group than control group, with statistically significant
difference, signaling to a greater predisposition of overnutrition patients to development of FISS.
Further investigations should be undertaken to elucidate correlation between overweight and FISS.
Perhaps the accumulation of adipose tissue in feline patients may predispose them to present FISS,
since it is know the correlation between inflammation and sarcoma genesis.
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2 WVCC 2012 - 145 –

Relationship between IL-8 and Slug in proliferation and survival of canine hemangiosarcoma cell

Jong-Hyuk, Kim
(Jong-Hyuk Kim, Milcah C Scott, Aric M Frantz, Daisuke Ito, Ashley J Graef et al)

Masonic cancer center, University of Minnesota, Minneapolis 55455, USA

[email protected]

Introduction
Canine hemangiosarcoma (HSA), an extremely common neoplasm in dogs is an aggressive, highly
metastatic and incurable tumor. However, biological and pathological features of canine HSA are
largely unknown and it is needed to develop an effective therapeutics. IL-8 is a proinflammatory
chemokine has been shown to play a role in tumor cell progression and survival, but the role has
not been elucidated in animal tumors. IL-8 gene was specifically up-regulated in HSA tissues and
cell lines compared to hematoma samples, non-malignant lesion in our previous studies with
microarray data.

Materials
To determine the function of IL-8 in proliferation of tumor cells, we investigated IL-8 mRNA and
protein expression in four HSA cell lines grown between normal HSA medium and no growth factor
medium, but the results were not consistent in different cell lines. Since canine HSA is
heterogeneous and IL-8 is likely to play differently depend on cell line, we tried to find other factors
associated with the role of IL-8.

Results
From the microarray data, it revealed that Snail family gene which not only acts primarily as a key
inducer of epithelial-mesenchymal transition but also plays a role in cell survival, in particular, Slug
was abundantly expressed in most cell lines and there was a reverse relationship between Slug and
IL-8 gene expression in cell lines from spleen HSA, but not non-spleen HSA.

Conclusions
Verifying the correlation between IL-8 and Slug could lead to uncover the function of them in
proliferation and survival of HSA cells.
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A survey to detect possible environmental factors associated with the occurrence of canine
lymphomas.

Danielle Almeida Zanini

(Danielle Almeida Zanini, Adriana Tomoko Nishiya, Katia Cristina Kimura,Rodrigo Ubukata,Ricardo
Augusto Dias and Maria Lúcia Zaidan Dagli)

Department of Clinics, School of Veterinary Medicine, University Anhembi Morumbi, São Paulo,
Brazil., São Paulo, Brazil

[email protected]

Introduction
Lymphomas are hematopoietic malignancies originating from lymphoid cells. According to
literature, lymphoma in dogs is similar to non-Hodgkins lymphoma in humans. Some studies show
that environmental factors increase the risk of developing lymphoma. This work aims to investigate
the correlation between exposure to household environmental tobacco smoke (ETS) and other
environmental factors which could increase the risk of canine malignant lymphoma.

Materials
A case-control study involving 83 dogs with malignant lymphoma and 84 controls was performed.
The owners of all animals were interviewed through an epidemiological questionnaire that sought
information about possible environmental contaminants such as passive smoking, contact with
pesticides and herbicides and contaminants physical, chemical and biological water, air and earth.

Results
Results have shown that purebred dogs presented a higher number of representatives with
malignant lymphoma; multicentric form and stage 4 were the most observed. Any gender
preference for the development of lymphoma has been observed. Dogs over 10 kg, which are
permanently kept outdoor and near busy streets or avenues (10 to 20 thousand vehicles and 1 to
1.3 thousand motorcycle per hour) had a higher risk for development of the disease (OR: 3.1, 95%
CI: 1.4 – 6.9, p = 0,005). There was no association with environmental tobacco smoke (ETS).

Conclusions
These results suggest that environmental factors, such as air pollution caused by vehicles, may
increase the risk of developing canine malignant lymphoma. These results also reinforce the need
to control air pollutants in the environment.
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Immunohistochemical detection of progesterone and cell proliferation in canine mammary

tumours

Julianna Thuróczy
(Julianna Thuróczy, Edina Perge, Eszter Kollár, Linda Müller and Lajos Balogh)

SzIU, Faculty of Veterinary Science Budapest, Budapest, Hungary

[email protected]

Introduction
The presence of progesterone and its correlation with cell proliferation were studied by
immunohistochemistry from 43 canine mammary tumours, using monoclonal antibodies against
progesterone, produced for use in a quantitative ELISA for serum progesterone determination, and
nuclear antigen Ki-67.

Materials
Positive cells were visualised by nuclear or cytoplasmic staining and counted per 500 cells.

Results
Benign tumours showed high progesterone positivity (196.42 ± 25.91) and low cell proliferation
rate (52.14 ± 16.73), malignant tumours had low numbers of progesterone-positive cells (68.19 ±
17.53) and revealed higher cell proliferation rates (141.72 ± 23.65), the difference was statistically
significant (p<0.005. The difference of the number of progesterone-positive and Ki-67-positive cells
found in malignant and benign tumours showed a negative correlation of medium degree with the
number of progesterone-positive cells. The number of Ki-67-positive cells was more than twice as
high (2.32 ± 0.66) as that of the progesterone-positive cells in the malignant tumours. The
proportion of progesterone-containing cells was 13.63 ± 3.5 % in malignant tumours and 39.28 ±
5.18 % in benign tumours. The overall proportion of progesterone-containing cells was 17.81 ± 7.93
%.

Conclusions
The majority of progesterone receptors are present in unliganded form. The progression towards
malignancy in spontaneous mammary tumours is associated with a decrease in hormonal steroid
dependency. The presence of progesterone in the cytoplasm of tumour cells is thought to indicate
the existence of a self-healing mechanism of the organism which is supported by the significantly
higher progesterone content found in the cytoplasm of benign tumour cells.
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Epidemiological study of the prevalence of canine mammary gland tumor in a Veterinary Hospital

Michele S. Frehse
(Michele S. Frehse, Maria Isabel Mello Martins, Marcos Cesar Sant Anna, Ana Paula Bracarense,
Elisângela Olegário da Silva, et al)

Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina - UEL,PR,

Brazil, Londrina, Brazil

[email protected]

Introduction
Mammary tumors are the main reasons to death in bitches, early detection and preventive
statement are crucial for survival of affected dogs. The aim of this study was to determine the
prevalence of canine mammary tumor (CMT) of the Department of Theriogenology, Veterinary
Teaching Hospital-Universidade Estadual de Londrina (UEL), Paraná, Brazil, during 2006 to 2010.

Materials
The data survey was obtained by medical records and a form where reviewed aspects of age,
breed, clinical sign, history, reproduction, metastasis and histopathological findings was conducted.
The form data were inserted to a statistic program EPI6 (CDC-Atlanta) to calculate the frequency
and tables. Statistical test of chi-square and Fischer with significance of 5% was used.

Results
During 2006-2010, 199 bitches had mammary neoplasia diagnosis. Thirty five percent was mongrel,
and 65,3% classified in breeds. Mean age was 8 yr-old (58,2%). The median size of tumor 4 -10 cm
corresponding 53,8%. Only 25,4% presented single tumors and 74,6% multiple. Majority 86,4%
were malignant and 13,6% benign. Breed, age, nutritional status, castration, hormonal
administration, reproductive history, tumor size and ulcerated tumors did not associate to the
malignity (p&gt;0,05). Multiples tumors were associated to the malignity (p=0,026). The malignant
neoplasia was associated to lymph node metastasis (histopathology diagnosis p=0,047) but not to
pulmonar metastasis.

Conclusions
This study demonstrates that the prevalence of CMT was high and has significance in the studied
region. The histopathology pattern of mammary tumors of medium, multiple and malignant type
were more frequent. The association between malignity and lymph node metastasis was observed.
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One-stage costal resection and elbow fold flap cutaneous reconstruction in three cats with
thoracic wall soft tissue sarcoma

Melissa Pottier-Drouard
(Melissa Pottier-Drouard, François Serres, Laurent Marescaux, Dominique Tierny and Antoine
Hidalgo)

ONCOVET, Villeneuve d’ascq, France

[email protected]

Introduction
Soft tissue sarcomas in cats have been of growing interest since the 1980’s. First recognized as
inflammation related phenomena, they have been considered as a complication of sub-cutaneous
vaccination. Tumors involving thoracic wall can be challenging to treat because of the soft tissue
defect resulting from superficial resection. Most of the time in such cases, latissimus dorsi muscle
can not be preserved. Cutaneous reconstruction can be achieved through local flap, thoraco-dorsal
or elbow fold flap. We describe here the use of the latter flap in three cases.

Materials
Three cats were presented for soft tissue sarcomas with thoracic wall involvement. All cases were
treated by costal and latissimus dorsi resection and by radiotherapy. Reconstruction was achieved
either by a combination of polypropylen mesh and omentalization or by muscular transposition
followed by a cutaneous flap originated from the elbow fold. In one cat, brachytherapy catheters
were implanted at the time of reconstruction.

Results
No surgical complications was observed. One case of wound dehiscence was observed at two
weeks, probably caused by postoperative radiotherapy. Margins were clean in the three cases, and
no local recurrence was observed at two years, one year and six months.

Conclusions
Basic management of feline soft tissue sarcoma includes as in other species such dog and human
being wide resection followed by radiotherapy. Thoracic wall sarcomas can be more difficult to
treat as surgical management can be challenging. In these cases, wide resection was easily achieved
using conventional thoracic wall reconstruction methods and elbow fold flap.
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Immunohistochemical Detection of Estrogen Receptor Alpha (ERα) in canine mammary tumors

and its correlation with prognosis

Luiz Roberto Biondi

(Luiz Roberto Biondi, Luciana Boffoni Gentile , Daniel Soares Sanches, Marguiti Isaura Soares and
Maria Lucia Zaidan Dagli)

School of Veterinary Medicine and Animal Science University of Sao Paulo, Brazil

[email protected]

Introduction
Immunohistochemistry has an important role in the assessment of prognostic and predictive
factors in invasive breast cancer (IBC) nowadays. ERα, a nuclear transcription factor activated by
estrogen that regulate growth and differentiation of normal breast epithelial cells, remain
functional at varying degrees in IBCs. In humans, breast cancers that express ERα are well-
differentiated and of low aggressiveness, denoting good prognosis, however this is still
controversial in dogs.

Materials
Thirty-two paraffin embedded samples of epithelial canine mammary tumors from Veterinary
School of Santos Metropolitan University, underwent immunolabelling for ERα. Stained slides were
submitted to data acquisition system and image analyzer software. Were considered ERα+ the cells
that showed brown stained nucleus with the percentage of positive cells graded as 0 = 75%.

Results
The histological diagnosis included 3 adenomas, 11 complex carcinomas, 11 simple carcinomas, 5
mixed type carcinomas, 1 carcinoma-malignant myoepithelioma and 1 comedocarcinoma. All
adenomas showed positive score for ERα while 18 carcinomas were reactive and 9 were negatives.
The attempt to correlate the ERα score with histological type and grading, age and tumor diameter
showed no statistically significant difference (p> 0.05) in linear and multinomial regression models.

Conclusions
Although there was an important difference among benign and malign lesions scores, malignant
tumors did not show any association between score, histological type or grade demonstrating that,
unlike woman breast cancer, estrogen receptor is not a marker of degree of tumor differentiation
on canine breast cancer.
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Prognostic of mast cell tumor based on clinical parameters

Thaís Andrade Costa Casagrande

(Thaís Andrade Costa Casagrande, Thais Rodrigues Macedo, Samanta Rios Melo,
Carolina Scarpa Carneiro, Maria Lúcia Zaidan Dagli, Julia Maria Matera, and Robson Fernando
Pasquale Silva)

Positivo University, Curitiba, Brazil

[email protected]

Introduction
The clinical parameters have been used to support prognostication of canine mast cell tumors.
However there are little studies that could bring convergent information. This is a prospective
clinical study, those objectives to identify clinical markers of prognosis for canine MCT.

Materials & Methods

Were included 81 dogs with histopathological diagnosis of MCT that were monitored for at least
one year after surgery. They were submitted to clinical evaluation, including historical and physical
examination and information related to neoplasia, surgery and chemotherapy. Acceptable
confidence interval was 95%.

Results
The variables age (p=0.001), consistency (p=0,018), growth velocity (p=0,028), bleeding (p=0,031),
surface form (p=0,029), adherence to deep plans (p=0,001), erythema (p=0,035), ulceration
(p=0,011), temperature (p<0,0001), clinical staging (p=0,011), metastasis (p<0,0001), systemic signs
(p=0,009), histological grade (p=0,002) showed an association with the death rate and neoplasm
free time by log-rank test. In multivariate analysis, multiple Cox, irregular surface, presence of
bleeding, metastasis and systemic signs were presented as independent variable.

Conclusion
The correspondence of the anamnesis data and clinical exam with the prognosis showed a profit
result.
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