validation of immunocytochemistry in canine thyroid … · 2017-08-04 · or sedation. therefore,...

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

VALIDATION OF IMMUNOCYTOCHEMISTRY

IN CANINE THYROID TUMORS

By

Hermans Leen

Promotor: Prof. Dr. S. Daminet Research Report

Co-promotor: Prof. Dr. R. Ducatelle as part of the Master's Dissertation

© 2017 Hermans Leen

Disclaimer

Universiteit Gent, its employees and/or students, give no warranty that the information provided in

this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result

in any infringement of third-party rights.

Universiteit Gent, its employees and/or students do not accept any liability or responsibility for

any use which may be made of the content or information given in the thesis, nor for any reliance

which may be placed on any advice or information provided in this thesis.

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

VALIDATION OF IMMUNOCYTOCHEMISTRY

IN CANINE THYROID TUMORS

By

Hermans Leen

Promotor: Prof. Dr. S. Daminet Research Report

Co-promotor: Prof. Dr. R. Ducatelle as part of the Master's Dissertation

© 2017 Hermans Leen

Preface

In this manner, I would like to express my sincere gratitude to all people who contributed to this Master’s

Dissertation and without whom this work would not have been possible.

First of all I want to profoundly thank my Promotor, Prof. Dr. S. Daminet, for accepting my request to

write my Dissertation in the interesting field of veterinary internal medicine. Her knowledge and

commitment instigated my interest for research. Always ready for meetings and willing to help

overcoming scientific and practical obstacles, she made good collaboration possible between all people

involved in this project. Moreover I want to thank her for reading and correcting manuscripts of this work

and for her critical, but constructive feedback.

Further, I want express my thankfulness to my Co-promotor, Prof. Dr. R. Ducatelle, who was always

ready to discuss scientific solutions during practical work and to review staining outcomes. His ever

critical, but open view encouraged me to widen my own mindset.

Subsequently, two people deserve my deepest gratefulness for all the work they carried out in regard

to this project. Dr. Sofie Marynissen, thank you so much for all the time you spent collecting samples.

On top of your busy schedule, you were always willing to look for patients and to take samples. Moreover

you never hesitated to answer my emails, to inform me about your work or to show me practical

techniques. Also many thanks to Marjan Steppe for executing staining of the slides. You were always

available to inform me about staining protocols and staining results and you were always ready to explain

executed protocols. Furthermore, I really appreciate your ever quick and clarifying responses to my

emails.

Last but not least, I want to thank some people who supported me during this six years of study. I want

to start to express my thankfulness to my parents for giving me the freedom and support to chase my

dreams. Special thanks also to my sister, who was always ready to listen to my stories and encourage

me during the tough times. Finally, I want to thank my friends for being part of the sweet memories that

I made during my time in Ghent and that I will never forget.

Table of contents

PREFACE

TABLE OF CONTENTS

ABSTRACT ................................................................................................................................................................................ 1

SAMENVATTING ....................................................................................................................................................................... 2

INTRODUCTION ......................................................................................................................................................................... 5

Anatomy and physiology of the thyroid gland .................................................................................. 5

Thyroid masses ............................................................................................................................... 6

Introduction to used methods .......................................................................................................... 6

CHAPTER 1. CANINE THYROID TUMORS ............................................................................................................................... 8

1.1 TYPES AND PREVALENCE .................................................................................................................. 8

Signalment ....................................................................................................................................... 8

Histological subtypes ....................................................................................................................... 8

1.2 DIAGNOSIS AND STAGING .................................................................................................................. 9

Clinical signs .................................................................................................................................... 9

Thyroid hormone status ................................................................................................................... 9

Staging ............................................................................................................................................. 9

1.3 TREATMENT OPTIONS AND PROGNOSIS ............................................................................................ 11

CHAPTER 2. USE OF IMMUNOHISTOCHEMISTRY (IHC) AND IMMUNOCYTOCHEMISTRY (ICC) IN VETERINARY

MEDICINE ................................................................................................................................................................................ 13

2.1 DEVELOPMENT OF THE TECHNIQUE ................................................................................................. 13

2.2 INFECTIOUS AGENTS ....................................................................................................................... 14

3.3 MALIGNANCIES............................................................................................................................... 14

CHAPTER 3. MOLECULAR MARKERS IN CANINE THYROID TUMORS ............................................................................... 16

3.1 MOLECULAR MARKERS FOR PROGNOSIS .......................................................................................... 16

Differentiating follicular and medullary thyroid tumors................................................................... 16

Cellular proliferation markers ......................................................................................................... 16

3.2 MOLECULAR MARKERS AS POTENTIAL THERAPEUTIC TARGETS .......................................................... 17

Cyclooxygenase-2 ......................................................................................................................... 17

Vascular endothelial growth factor ................................................................................................ 18

P-glycoprotein ................................................................................................................................ 19

CHAPTER 4. AIMS ................................................................................................................................................................... 21

CHAPTER 5. MATERIAL AND METHODS .............................................................................................................................. 22

5.1 CASE SELECTION AND SELECTION OF CONTROL SAMPLES ................................................................. 22

5.2 CYTOLOGICAL SPECIMENS AND SAMPLE PREPARATION ..................................................................... 22

Thyroid carcinomas ....................................................................................................................... 22

Control samples ............................................................................................................................. 22

5.3 IMMUNOCYTOLOGICAL ANALYSIS ..................................................................................................... 24

Thyroglobulin ................................................................................................................................. 24

Calcitonin ....................................................................................................................................... 24

COX-2 ............................................................................................................................................ 24

VEGF ............................................................................................................................................. 25

CD-3 .............................................................................................................................................. 25

CHAPTER 6. RESULTS ........................................................................................................................................................... 26

6.1 THYROID TUMOR MOLECULAR MARKERS .......................................................................................... 26

6.2 LYMPHOID MARKERS ...................................................................................................................... 26

DISCUSSION ............................................................................................................................................................................ 29

Sample preparation protocols ........................................................................................................ 29

Positive control samples ................................................................................................................ 30

Role of molecular markers in thyroid carcinoma management ..................................................... 31

Future directions ............................................................................................................................ 32

REFERENCES ......................................................................................................................................................................... 33

1

Abstract

Thyroid masses represent 1,2-3,8% of all canine neoplasms and are therefore the most common form

of endocrine neoplasia [1, 2]. Up to 90% of these tumors are malignant, resulting in evidence of

metastatic disease in 16-38% of the cases at the time of diagnosis [2]. Therefore, Campos et al.

investigated prognostic factors and therapeutic targets in these tumors and validated

immunohistochemistry for prognostic and therapeutic molecular markers [3, 4]. Despite the promising

results found by Campos et al., a major drawback is that thyroid biopsies, needed for performing

immunohistochemistry, are not always available. In contrast, cytology is widely used in companion

animal medicine, as it is quick, less invasive, associated with lower cost and does not require anesthesia

or sedation. Therefore, the aim of this Master’s Dissertation was to validate immunocytochemistry of

molecular markers in canine thyroid tumors, based on fine-needle aspirate samples. Positive control

samples were used to evaluate the used protocol: thyroid smears for thyroglobulin and calcitonin, liver

tissue for P-glycoprotein and kidney smears for cyclooxygenase 2. However, staining of positive control

samples remained repeatedly negative, also after dilution series and antigen retrieval techniques.

Therefore we decided to review the effect of fixation technique, storage temperature and storage time

to analysis on staining outcome by using CD3 staining on lymphoid tissue smears. Despite poor sample

quality we could conclude that acetone fixation leads to a lot of cell loss during staining process and that

storing samples by -20 or even -70°C instead of room temperature can enhance staining outcome.

2

Samenvatting

Massa’s van de schildklier vertegenwoordigen bij de hond tussen de 1,2 en 3,8% van alle tumoren en

zijn daarmee de meest voorkomende tumoren van endocriene oorsprong [1, 2]. Tot 90% van de

schildkliermassa’s zijn maligne [2] en zowel de folliculaire cellen als de para-folliculaire cellen (of

C-cellen) van de schildklier kunnen tumoraal ontaarden, resulterend in respectievelijk folliculair

thyroidcarcinoma (FTC) of medullair thyroidcarcinoma (MTC) [1].

Gezien de kwaadaardige biologische natuur van de meeste schildkliertumoren, worden in 16 tot 38%

van de gevallen metastasen vastgesteld op het moment van de diagnose [2]. De mogelijke

behandelingsopties bij honden met thyroidcarcinoma zijn onder meer afhankelijk van de grootte van de

tumor, de mate van invasie in omliggende structuren, de beweeglijkheid van de tumor en het al dan niet

aanwezig zijn van metastasen [1]. Bij patiënten met niet-invasieve, beweeglijke tumoren zonder

metastasen geniet chirurgische excisie van de tumor de voorkeur [5]. Echter, bij de helft van de patiënten

die chirurgisch behandeld worden, wordt lokaal herval of optreden van metastasen vastgesteld binnen

de 2 jaar na de chirurgische ingreep [4, 6]. Vaak is er, omwille van de invasiviteit van de tumor, geen

chirurgie mogelijk en dan kan geopteerd worden om te behandelen met radiatie-therapie of therapie met

radioactief jodium (131I) [7-10]. Indien metastasen aanwezig zijn of de kans hierop zeer groot is, kan de

therapie (chirurgie of radiatie) aangevuld worden door chemotherapie met cisplatine of doxorubicine,

hoewel een stijging van de overlevingstijd met chemotherapie tot nog toe niet werd aangetoond

[1, 9, 11].

Gezien de resultaten van de behandeling van schildkliercarcinomen wisselend zijn, voerden Campos et

al. recentelijk een onderzoek uit naar verschillende prognostische parameters [3]. Uit dit onderzoek kon

geconcludeerd worden dat de diameter van de tumor, het volume, de expressie van Ki-67, ectopische

lokalisatie en folliculaire oorsprong predisponerende factoren zijn voor lokale invasiviteit van de tumor.

De aanwezigheid van metastasen was dan weer gecorreleerd met de diameter en het volume van de

tumor en met bilaterale lokalisatie van het carcinoom [3]. Na deze studie betreffende prognostische

factoren, onderzocht de groep ook de immunohistochemische (IHC) expressie van mogelijke

therapeutische targets door thyroidcarcinomen [4]. Vasculaire endotheliale groeifactor (VEGF) bleek tot

expresie te komen in 85% van alle onderzochte schildkliercarcinomen (zowel FTC als MTC);

cyclooxygenase 2 (cox-2) en P-glycoproteïne (P-gp) kwamen vooral tot expressie in de tumoren van

C-cel origine (MTC) [4]. Deze resultaten tonen aan dat VEGF een belangrijke therapeutische target kan

zijn bij zowel FTC als MTC en dat cox-2 en P-gp interessante targets zijn bij MTC. Deze bevindingen

zouden mogelijks een doorbraak kunnen betekenen in de behandeling van schildkliercarcinomen,

voornamelijk wanneer chirurgische excisie geen optie is [4].

Hoewel de resultaten veelbelovend zijn, dienen deze gevalideerde IHC kleuringen uitgevoerd te worden

op biopsieën. Deze zijn helaas vaak niet beschikbaar gezien schildkliertumoren steeds sterk

gevasculariseerd zijn. Percutane biopsieën zijn dus risicovol en worden daarom bijna nooit genomen.

Bijgevolg zijn biopsieën niet beschikbaar als er geen thyroïdectomie wordt uitgevoerd, bijvoorbeeld als

er ook metastasen zijn vastgesteld of wanneer eigenaars geen chirurgie wensen [1].

3

Cytologie daarentegen is een frequent aangewende techniek in de diergeneeskunde omdat het snel is,

weinig invasief, relatief goedkoop en bovendien geen sedatie of anesthesie vereist. Fijne naald

aspiraten (FNA) van schildkliercarcinomen zouden dan ook vele voordelen kunnen bieden vergeleken

met biopsieën [12]. Daarom was het oogmerk van deze scriptie om de door Campos et al.

immunohistochemisch gevalideerde merkers ook immunocytochemisch (ICC) te valideren op

cytologische preparaten van FNAs van schildkliercarcinomen. Het doel was om de voorbereiding van

cytologische slides te standaardiseren en ICC kleuringen te valideren met het oog op het onderscheiden

van FTC van MTC op basis van calcitonine en thyroglobuline. Bovendien wilden we de ICC kleuring van

de moleculaire therapeutische merkers (cox-2, P-gp en VEGF) valideren.

Echter, extrapolatie van IHC gevalideerde technieken naar ICC is technisch uitdagend omwille van de

grote verschillen in de aard van de preparaten. Om de resultaten van de gebruikte technieken te

evalueren, maakten we gebruik van positieve controle stalen. Voor thyroglobuline, calcitonine en VEGF

werden uitstrijkjes van FNAs van de schildklier gebruikt, voor P-gp leverweefsel en voor cox-2

nierweefsel. De FNA stalen werden zo snel mogelijk na het overlijden (< 12 uur) genomen en meteen

gefixeerd in methanol. Daarna werden de standaard kleuringen voor P-gp, cox-2, VEGF en calcitonine

via de Dako Autostainer+ uitgevoerd. De eerste resultaten vertoonden echter zeer weinig specifieke

kleuring en veel achtergrondkleuring was aanwezig. Bovendien konden we vaststellen dat er slechts

weinig cellen op het preparaat aanwezig waren. Daarna werd er getracht de resultaten van de

kleuringen te verbeteren door antistof-verdunningsreeksen aan te leggen (voor P-gp, cox-2, VEGF en

calcitonine) en een antigen retrieval stap uit te voeren bij de kleuring van nierweefsel voor cox-2. Echter,

geen van deze technische ingrepen was in staat om het resultaat van de kleuringen van de positieve

controles te verbeteren.

Cytologische preparaten zijn echter veel delicater dan de coupes die gebruikt worden voor IHC en

verschillende factoren kunnen de resultaten van de kleuringen beïnvloeden. Daarom werd besloten om

het effect van de fixatie techniek, de bewaartemperatuur en bewaartijd tot analyse op de resultaten te

onderzoeken. Hiervoor werd gebruik gemaakt van cytologische preparaten van lymfoïd weefsel

(genomen via FNA uit de Lymphonodus Popliteus) die gekleurd werden voor CD3. Deze merker werd

gekozen als controlemerker omdat de antistof tegen CD3 gekend is om goede kleuringen te geven, ook

in minder ideale omstandigheden [12-14]. Echter, nadat we de eerste kleuringen hadden uitgevoerd,

bleek dat het overgrote deel van de cellen op de slides vetcellen waren in plaats van lymfoïde cellen.

Daardoor was het moeilijk om het effect van de verschillende protocollen op de kleuringen te

beoordelen. Niettemin kon worden vastgesteld dat preparaten gefixeerd met aceton gedurende de

procedure veel cellen verliezen, wat resulteert in een lager diagnostisch potentieel. Wanneer de fixatie

daarentegen werd uitgevoerd met formaline, bleven de cellen beter aan de slides kleven. Deze

resultaten zijn compatibel met eerder gepubliceerde onderzoeken [15]. Het nadeel aan formaline fixatie

is echter dat er een extra antigen retrieval stap moet worden toegevoegd aan de procedure. Bovendien

zullen zelfs na die antigen retrieval stap niet alle antistoffen het antigen herkennen [15]. Of formaline

fixatie al dan niet gebruikt kan worden, hangt dus af van het type weefsel en het antigen dat men wenst

aan te kleuren. Daarnaast bleken preparaten die bij -20°C bewaard werden in plaats van op

kamertemperatuur ook betere resultaten te geven.

4

Om ICC van moleculaire merkers bij schildkliertumoren in de toekomst te valideren, zal nog meer

onderzoek nodig zijn. Gezien de verschillende eigenschappen van elk weefsel en elke antistof/antigen,

zal voor elk antigen individueel het best mogelijke protocol moeten worden opgesteld. Echter, Diff-Quik

kleuring van genomen stalen, zou kunnen helpen om de kwaliteit van de stalen te beoordelen voor het

uitvoeren van de ICC kleuring. Ook de positieve controles kunnen nog geoptimaliseerd worden. Zo is

er in ontstekingsweefsel bijvoorbeeld een veel sterkere expressie van cox-2 dan in nierweefsel, en

zouden FNAs van ontstekingsweefsel dus betere positieve controles voor de cox-2 kleuring kunnen

opleveren.

5

Introduction

Anatomy and physiology of the thyroid gland

The thyroid gland is a bilobated endocrine gland located adjacent to the lateral surface of the trachea

under the larynx [16]. It harbors two main cell types: the follicular cells or thyrocytes and the parafollicular

or C-cells. Follicular cells, when stimulated by thyroglobulin, produce the thyroid hormones

triiodothyronine (T3) and thyroxin (T4) out of iodine and tyrosine [17]. These hormones play a critical

role in the regulation of numerous key metabolic pathways, energy homeostasis and are essential for

the adequate development and differentiation of all cells in the body [18, 19]. In between these follicular

cells, parafollicular cells are located in the connective tissue. These cells have a neuroendocrine origin

and secrete calcitonin, which is an important hormone in calcium homeostasis as it inhibits absorption

of calcium by the intestine, reduces he resorption of bone by the osteoclasts and decreases the amount

of calcium reabsorption by renal tubular cells [17, 20]. Both of these cell types can undergo a malignant

transformation, which results in either follicular thyroid carcinoma (FTC) or medullary thyroid carcinoma

(MTC) respectively.

Figure 1. The anatomy of the thyroid and parathyroid glands in dogs.

(From: Miller's Anatomy of the Dog, 2013, [21])

6

Thyroid masses

Between species, prevalence and clinical significance of masses of the thyroid gland vary widely. In

humans, thyroid masses account for approximately 4% of all diagnosed cancers, making it the most

common human endocrine neoplasia [22]. Thyroid masses in humans are mostly benign; 42-77% are

non-neoplastic colloid nodules, 15-40% are adenomas and only 8-17% are carcinomas [23].

Radiation exposure to the thyroid gland in childhood, age, female sex, and family history are risk factors

that increase the incidence of well-differentiated thyroid cancer in humans [11].

Thyroid tumors in dogs are usually malignant and non-functional, with less than 25% of the dogs showing

clinical evidence of hyperthyroidism [1, 24, 25]. Hence, the clinical signs in dogs are mostly not provoked

by the hypersecretion of thyroid hormones, but instead are the result of impingement on surrounding

structures. The most common clinical signs in dogs thus include coughing, dysphagia, dyspnea and

dysphonia [2]. This is in strong contrast with thyroid tumors seen in cats, which are mostly benign and

functional [26].

For dogs with freely movable thyroid tumors with minimal invasion into surrounding structures and no

evidence of metastatic disease, surgical resection is the treatment of choice [7]. However, at time of

diagnosis as much as 50-75% of the dogs appear no good candidates for surgical therapy because of

invasiveness or metastatic disease [27]. Moreover, approximately half of the patients treated with

thyroidectomy experience local recurrence or metastatic disease within 2 years of surgery [4, 6].

Therefore, recently, Campos et al. investigated the expression of prognostic molecular markers in

canine thyroid carcinoma, concluding that tumor diameter, tumor volume, Ki-67 expression, ectopic

location and follicular cell origin were positively associated with local invasiveness and that tumor

diameter, tumor volume and bilateral location were positively associated with presence of distant

metastasis [3]. However this information can help in daily practice for treatment adaptation and risk

assessment of patients, no treatment was shown to be effective as adjunctive therapy [3].

Consequently, Campos et al. investigated the immunohistochemical (IHC) expression of potential

therapeutic targets in canine thyroid carcinoma [4]. This study showed that 85% of all examined thyroid

carcinomas exhibited a high percentage of vascular endothelial growth factor (VEGF) positive tumor

cells, indicating that VEGF may play an important role in the progression of canine thyroid cancer and

may be an interesting molecular target for the treatment of both FTC and MTC in dogs. In MTC,

expression of cyclooxygenase 2 (cox-2) and P-glycoprotein (P-gp) was common, suggesting that these

may represent valuable therapeutic targets in dogs that are not good surgical candidates. For example,

inhibition of P-gp could increase tumor sensitivity to chemotherapy and improve treatment outcome [4].

Introduction to used methods

Despite the promising results found by Campos et al., a major drawback is that thyroid biopsies, needed

for performing IHC, are not always available. Indeed, canine thyroid tumors are highly vascular and

therefore percutaneous biopsies are contraindicated [1]. Furthermore, surgical biopsies contain also a

high risk of hemorrhage and are consequently seldom performed [1]. Hence, biopsies are not available

in patients with invasive thyroid tumors, in patients with metastasis or when owners decline surgery.

7

In contrast, cytology is widely used in companion animal medicine, as it is quick, less invasive,

associated with lower cost and does not require anesthesia or sedation. Accordingly, fine-needle

aspirate (FNA) sampling can offer a lot of advantages compared to biopsies, nonetheless

immunocytochemic analysis of this samples includes some special concerns and often manual methods

or optimization of existing protocols for automatic staining are needed [12, 15].

An important problem that is frequently encountered in immunocytochemistry (ICC) is that cells are

washed away from the slides easily in automated methods, resulting in lack of diagnostic power.

However, it has been shown that different fixation methods can influence this phenomenon strongly. For

example, formalin fixation is known to enhance cell adhesion to the slide, whereas the standard acetone

fixation leads to a lot of cell loss during staining [12, 15]. In contrast, formalin fixation is at times

associated with loss of antigenicity, even after antigen retrieval techniques. The protocol followed prior

to staining is thus of great importance and may vary depending on the targeted antigen. The most

important factors that can affect staining outcome include type of fixation, sample storage temperature

and storage time to analysis [12, 28]. Lastly, also inactivity of some antibodies in ICC techniques must

be considered.

To validate ICC of molecular markers in canine thyroid tumors, the technique to stain antigens of interest

must be verified on positive control samples. During practical work in this Master’s Dissertation however,

positive control samples resulted repeatedly negative after staining. Therefore we decided to review

different sample preparation protocols, based on the review paper on ICC published by Priest et al. [12].

We evaluated the effects of fixation method, storage temperature and storage time to analysis on

staining outcome of cytological smears in order to standardize FNA sample preparation for ICC. We

opted to start with CD3 staining of lymphoid tissue, which is proved to be a reliable antibody with good

staining outcomes, also in unfavorable circumstances [12-14]. We aimed to elect the preparation

protocols giving the highest reproducible results on lymphoid tissue and apply these protocols on

positive control samples and thyroid carcinoma tissue. In this way we sought to improve staining

outcomes of positive control samples and eventually validate ICC staining of molecular markers in

thyroid carcinoma on FNA smears.

8

CHAPTER 1. Canine thyroid tumors

1.1 Types and prevalence

In dogs, thyroid masses only represent 1,2-3,8% of all neoplasms [1], still they are the most common

form of endocrine neoplasia [2]. Up to 90% of these thyroid tumors are carcinomas [29], which are

mostly growing fast and invasive into surrounding structures such as the trachea, larynx, jugular veins

and carotid sheath. This results in a biologically highly malignant character of the tumors with evidence

of metastatic disease in 16-38% of the cases at the time of diagnosis and even up to 80% at the time of

necropsy [2]. Even though adenomas have also been described, their prevalence is much lower and as

they do usually not cause any clinical symptoms, they mostly are an incidental finding at necropsy

[2, 6, 30].

Signalment

A study on breed predisposition toward canine thyroid carcinoma revealed that Golden retrievers,

Beagles and Siberian huskies are overrepresented [29]. Other reports show that also Boxers had a

significantly greater risk for thyroid carcinoma [31, 32]. Concerning age, most studies report ranges

from 5 to 18 years at presentation, with a median age of 9 to 10 years [2]. However, the majority

of dogs in a large retrospective study were older (between 10 and 15 years) [29]. Although there is a

strong female preponderance for thyroid cancer in humans [33], in dogs no gender predilection was

found [2, 29].

Histological subtypes

According to the World Health Organization, thyroid cancers can be classified into subtypes based on

histopathological characteristics. Human thyroid carcinomas are classified into 4 subtypes.

2 well-differentiated types: papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC),

both arising from the thyroid follicular cells, which account respectively for 80-85% and 10-15% of all

human thyroid cancers. The third type is the medullary thyroid carcinoma (MTC), which arises from the

C-cells and accounts for 5% of all human thyroid cancers. Lastly, the fourth type is the anaplastic thyroid

carcinoma (ATC), which is a rare, aggressive and lethal form, accounting for only 1% of all human

thyroid carcinomas [34].

Canine thyroid cancers on the other hand are classified as follicular (FTC) or medullary thyroid

carcinoma (MTC). Canine FTC have been further classified into well-differentiated, poorly-differentiated

and undifferentiated. Well-differentiated thyroid carcinomas (dFTC) are subdivided into follicular,

compact, follicular-compact and papillary thyroid carcinomas on the basis of predominant histologic

pattern [35, 36]. MTC are less common than FTC, however the prevalence of MTC is presumably

underestimated due to the fact that they are difficult to distinguish from compact dFTC by microscopic

evaluation. Therefore, IHC for calcitonin or for markers of neuroendocrine tissue is required for their

identification [24]. The distinction between this two types of thyroid carcinoma might be important

concerning prognosis, since MTC are thought to have a less malignant nature compared to other thyroid

carcinomas [1, 24]. However, recent studies do not support this hypothesis [2]. A recent study showed

9

that MTC were significantly less likely to be locally invasive at presentation, however no difference in

the incidence of metastatic disease was seen at time of diagnosis. Furthermore, the study showed that

there was no prognostic difference between MTC and FTC after surgical treatment [3].

1.2 Diagnosis and staging

Clinical signs

Mostly, dogs are presented because the owner’s discovery of a cervical mass [10, 24]. Differential

diagnoses for masses in this region include abscesses, granulomas, salivary mucoceles, other primary

tumors, lymphoma and nodal metastasis from head and neck tumors [1]. The time from the owner’s

recognition of the mass until diagnosis is 1-2 months [2]. In dogs with thyroid tumors clinical signs are

often related to the mass effect of the thyroid tumor and include coughing, dysphagia and dysphonia

[32]. Also dyspnea can be present, relating to upper airway disruption or to lower airway compromise in

case of pulmonary metastatic disease [2, 24]. In one clinicopathologic study, surgical exploration

revealed that neoplastic tissue was present in the lumen of the jugular vein in 6 of the 23 examined

dogs. However occluding thrombosis of major veins was rare. In one case, neoplastic tissue was present

in the jugular vein, cranial vena cava and right atrium, resulting in facio-cervical and foreleg edema [32].

Also Horner’s syndrome has been associated with a functional follicular thyroid carcinoma in a dog [37].

Definitive diagnosis can be achieved through histologic examination of a biopsy sample or after surgical

removal of the tumor [2]. Even tough in some cases cytology may be sufficient, diagnostic accuracy of

this technique is low because of frequent blood contamination [1, 38].

Thyroid hormone status

Most dogs with thyroid tumors are euthyroid and clinical signs of hyperthyroidism are, in contrast with

cats, absent in most dogs with thyroid tumors. Leav et al. [30] reported hyperthyroidism in around 20%

of the dogs with thyroid tumors in a study performed in the Netherlands, although most American studies

reported prevalences that were much lower; 0 to 6% [6, 32]. The reason for this apparent geographic

discrepancy is unclear [39]. However, a more recent study reported elevated T4 levels in 31% of the

dogs, although only two of them exhibited clinical signs of hyperthyroidism [10], such as polyuria,

polydipsia, polyphagia and weight loss [37, 39]. Furthermore, also a lowering of thyroid hormones is

mentioned in literature, which could be due to euthyroid sick syndrome, although clinical signs of

hypothyroidism are seldom seen [6, 10].

Staging

As in other neoplastic diseases, the World Health Organization (WHO) TNM-staging system is used to

stage patients with thyroid tumors (Table 1). Physical examination is important to determine the size

and degree of fixation of the tumor (often related to each other) and to assess the regional lymph nodes

for metastasis; including mandibular, parotid and medial retropharyngeal lymph nodes. On palpation,

thyroid adenomas are mostly freely moveable whereas thyroid carcinomas are either well circumscribed

and freely moveable (24-55%) or diffusely infiltrative and fixed (up to 67%) [1, 2, 5, 24, 32]. Evaluation

of invasiveness of the tumor may require sedation, because it can easily be overestimated in the awake

10

dog. Since the majority of dogs diagnosed with thyroid neoplasia are middle to older aged, it is important

to assess their general health with hematology, serum biochemistry and urinalysis. Furthermore, serum

thyroxine and TSH concentrations should be measured to assess their thyroid status [1]. Additionally,

medical imaging techniques can be used to gather more detailed information on the tumor’s

characteristics. Radiographs of the neck usually show a soft tissue mass and are useful to defect

deviation or compression of the trachea or larynx. Thoracic radiographs, taken in three directions, are

used to evaluate the presence of pulmonary metastasis. However, radiographs do not provide

information on vascularity or invasiveness [2]. Ultrasonography, on the other hand, provides a more

detailed image and can easily be used in daily veterinary practice as it is quick, non-invasive and

relatively low-priced. Moreover, it could perhaps be used to guide fine-needle aspirates for diagnostic

purposes [40], because the risk of significant hemorrhage is thought to be reduced in ultrasound guided

biopsies [2]. Furthermore, advanced imaging techniques, such as MRI and CT, are indicated for

planning radiation treatment in dogs with invasive or incompletely resected carcinomas [1]. Furthermore

these techniques are found to be very accurate and useful in preoperative diagnosis and staging of

patients. As indeed MRI can provide more sensitive information concerning the degree of invasiveness

of the thyroid mass and CT scans of the thorax are more accurate in diagnosing pulmonary metastasis

[2, 41]. Yet these techniques are not always used in common veterinary practice because

ultrasonography or surgical exploration might be preferred for their cost-effectiveness in determining

tumor invasiveness and resectability [1]. Also scintigraphy with either 99mtechnetium pertechnetate or

131iodine has also been described for the diagnosis and staging of dogs with thyroid tumors [6, 25]. In a

study, no relation was seen between distribution of 99mtechnetium pertechnetate uptake and histologic

diagnosis, but there appeared to be an association between distribution of uptake and capsular invasion;

as tumors with extensive capsular invasion had a poorly circumscribed uptake of pertechnetate [25].

Although scintigraphy is useful for

identifying malignant ectopic tissue,

which can be difficult to detect with

conventional imaging techniques,

and regional lymph node metastasis,

it did not appear to offer any

additional benefit for detection of

pulmonary metastases compared

with thoracic radiography [25]. The

major advantage of thyroid

scintigraphy in suspected thyroid

tumors is to assess iodine uptake.

Thereby confirming or not that the

patient is a candidate for I131

treatment (as a sole or as an adjunct

therapy) [39, 42].

Table 1. TNM classification of tumors in domestic animals

(Adapted from: Owen LN, ed. World Health organization, Geneva, 1980)

11

1.3 Treatment options and prognosis

As mentioned earlier, treatment options for dogs with thyroid carcinoma differ depending on size of the

tumor, degree of invasion, mobility of the tumor and presence of metastasis [1]. In dogs with mobile,

unilateral tumors and minimal invasion into surrounding structures, surgery is considered the best

therapeutic procedure [5]. In patients with fixed and invasive tumors that are not good candidates for

surgery, radiation therapy is effective for local control of the tumor [7, 8]. However regression is slow

and maximal reduction in tumor size can take 6 to 22 months, the response to therapy is long-lasting

[8, 9]. As in cats, where functional thyroid tumors are often treated with radioactive iodine, also in dogs

with thyroid carcinoma radioisotope iodine therapy is performed nowadays. Formerly, it was assumed

to be ineffective for the treatment of large tumors [9], however a study concluded that radioactive iodine

therapy can be effective at extending survival time in dogs with invasive thyroid tumors [10]. It can be

useful in cases where surgery alone is not likely to be curative (e.g. when complete surgical removal

could not be achieved) or in case of metastatic disease [10]. Several studies on the use of chemotherapy

have also indicated that doxorubicin or cisplatin may play a role in the management of patients with

thyroid carcinoma; reported response rates vary from 30-50%, however improved survival times could

not yet be obtained [43, 44]. Even though the role of chemotherapy in canine thyroid carcinoma has to

be further investigated, it should be recommended in patients with a high risk to development of

metastatic disease after performing surgery or radiation therapy [1, 9, 24].

Figure 2: Algorithm for the treatment of dogs with thyroid carcinoma

In dogs with mobile tumors and minimal invasion into surrounding structures, surgery is considered the best therapeutic

procedure. In patients with fixed and invasive tumors that are not good candidates for surgery, radiation therapy is effective

for local control of the tumor and also radioactive iodine therapy has been proven to extend survival time in dogs with invasive

thyroid tumors. Good local tumor control significantly decreases the risk of metastatic disease and is thus the most important

factor to contemplate in dogs without evidence of metastasis. In patients with evidence of metastasis, palliative treatment with

surgery, external beam radiation or chemotherapy can be performed.

(From: Liptak, J.M., Canine Thyroid Carcinoma. Clinical Techniques in Small Animal Practice, 2007 1)

12

As canine thyroid tumors are often biologically malignant tumors with evidence of metastatic disease in

16-38% of the patients at time of diagnosis [2], median survival time (MST) for dogs with thyroid

carcinoma left untreated is only 3 months [10]. However, with appropriate treatment, prognosis can be

improved [1]. The MST for dogs with mobile thyroid tumors without evidence of metastatic disease that

undergo surgical resection is more than 36 months [5]. Surgery in dogs with fixed thyroid carcinomas

lead to MST of only 10 months [24]. In these dogs survival time can be prolonged with radiation or

radioiodine-131 (131I) therapy. External beam radiation is associated with MST from 24,5 up to more

than 45 months [9, 42], whereas 131I therapy is associated with a MST of 28-30 months [10], when no

distant metastasis are present.

As in all malignant cancers, local recurrence of the tumor after surgical resection or development of

metastatic disease affects prognosis in dogs with thyroid carcinomas. Local recurrence or progression

is reported in up to 30% of the patients following thyroidectomy and in 24% of the patients that did

undergo radiation therapy [1, 9, 45]. Good local tumor control also significantly decreases the risk of

metastatic disease [9], whereas tumor volume greater than 20 cm3, tumor diameter greater than 5 cm

[30], bilateral thyroid tumors [9] and invasion of the tumor into cervical vascular structures [6] are

negative prognostic markers as they are more often associated with metastasis.

13

CHAPTER 2. Use of immunohistochemistry (IHC) and

immunocytochemistry (ICC) in veterinary medicine

2.1 Development of the technique

The concept of immunochemistry is based on the detection of a target using an antibody and subsequent

visualization using a chemical reaction to produce a color change. In 1941, the concept of

immunohistochemistry (IHC), where the detection of the molecular target takes place in a fixed tissue,

was performed for the first time [46, 47] and thereafter subsequent refinements in the procedure were

developed [48]. This technical improvements (e.g. antigen retrieval techniques), together with the

increasing range of available antibodies, enhanced sensitivity and made that IHC is now widely used in

human and veterinary medicine for diagnosis of diseases associated with autoantibody deposition,

infectious diseases and for identification and typing of malignancies, as well as in modern research

[47, 49]. Indeed, the number of immunohistochemical tests offered by veterinary diagnostic laboratories

for the diagnosis of infectious and neoplastic diseases has increased vastly in the last decade [50].

However it is a widely used technique, biopsies that are needed to perform IHC are not always available.

For example in patients whose condition may make them unavailable for surgical biopsies, the use of

IHC is excluded. Therefore, the use of immunocytochemistry (ICC), where the detection of the target

takes place in cells on cytological smears, has increased rapidly over the last decades [28]. Indeed,

FNAs are less invasive and quicker, however important information concerning histologic architecture

is limited [51]. Still, ICC has contributed to an increased diagnostic accuracy of FNAs and is often used

as an adjunct to cytological examination, especially in the diagnosis of human neoplasia [52].

Figure 3: Schematic overview of the principle of IHC

After fixation and quenching of endogenous peroxidase,

primary antibodies are added to the slide and bind onto the

antigen of interest present in the tissue. Afterwards,

Biotin-labelled secondary antibodies from another animal

species that recognize the Fc-chain of the primary

antibodies are added and multiply the signal. Then,

streptavidin-peroxidase is added to the slide and binds with

the present biotin. When thereafter DAB is added, the

peroxidase enzyme oxidizes DAB which will turn into a

brown pigment that precipitates as a brown solid located at

the site of the antigen of interest. This brown solid pigment

can then be examined by microscopic evaluation [53].

This principle is also valid in ICC.

(From: Magub, S. Immunohistochemistry: getting the stain

you want, 2016, 53)

14

2.2 Infectious agents

IHC is a rapid and reliable technique to detect a wide array of disease causing agents, including those

that are nonviable or difficult to isolate or culture [47]. Demonstration of an antigen in a lesion by IHC

techniques is indeed an important contribution to diagnosis, either at the time of investigation or

retrospectively [54]. Furthermore, IHC allows co-localization of an antigen with a morphological lesion,

which increases diagnostic accuracy and can help to elucidate pathogenesis [50]. In most cases,

sensitivity and specificity of IHC are good and IHC is even used as gold standard for some diseases

[55]. In a conference in 2002 on feline coronavirus and Feline Infectious Peritonitis (FIP), positive

immunohistochemical staining for viral proteins of macrophages within lesions in tissues taken by biopsy

or necropsy was considered the most definitive test for FIP [56]. Also a study on diagnosis of canine

distemper (CDV) encephalitis concluded that however inclusion bodies were a good diagnostic criterion

for the confirmation of CDV infection, the immunohistochemical demonstration of CDV antigen proved

to be superior. The study showed that CDV antigen was more prevalent than inclusion bodies in the

investigated tissue sections and moreover much more easily detectable [57]. Also in post-mortal

diagnosis of canine parvovirus IHC can be used to determine cause of death [58]. Also in human

medicine, ICC is used for the detection of various infectious agents [28, 59]. For example, the

identification of Mycobacterium tuberculosis in cytological specimens can be made using a monoclonal

antibody MTSS [60].

3.3 Malignancies

Also in oncological pathology, IHC is widely used since several years as an adjunct to light microscopy

in the diagnosis of various neoplasms [61]. Routine pathological analysis of tissue samples is based

upon the morphological aspects assessed by light microscopy. However, in some cases, diagnosis of

malignancies based on histopathology alone is challenging. In particular, investigation of the expression

of cell surface or intracellular markers by IHC is been of great use to determine the tissue of origin of

the neoplastic cells [62].

For example in canine round cell tumors, IHC staining for specific markers can be very useful since

different round cell tumors may have a similar morphologic appearance [63]. However, an accurate

diagnosis is important for determining prognosis and treatment in these tumors. In a study, staining

cutaneous round cell tumors for CD3 and CD79 (for lymphocytes), CD18 and MHC-II (for macrophages)

and tryptase (for mast cells) could elucidate the cell of origin in most cases [61]. Also in amelanotic

canine melanomas, IHC is used to differentiated them from carcinomas, sarcomas and round cell

neoplasms which differ in prognosis and treatment [64, 65]. In a study a staining cocktail containing

antibodies against PNL2, Melan-A, TRP-1 and TRP-2 was concluded to be cost-effective and efficient

in identifying canine oral amelanotic melanocytic neoplasms [64].

Furthermore, immunohistochemical expression of biomarkers can be used to assess prognosis in

malignant neoplasms. In malignant mammary tumors, the expression of urokinase plasminogen

activator (uPA) is significantly higher than in benign tumors and is associated with larger tumor size,

high Ki-67 expression, invasive growth, high histological grade, regional lymph node metastases,

15

development of distant metastases, lower overall survival (OS) and disease-free survival (DFS). Hence,

immunohistochemical expression of uPA is considered as a useful prognostic factor in dogs with

malignant mammary tumors [66]. Also expression of MMP-9 and Ki-67 are used in this tumor types as

prognostic factors [67].

Moreover, some of this biological markers can be exploited as therapeutic markers. The expression of

potential interesting therapeutic targets, such as VEGF or Cox-2, by malignant cells can be assessed

by IHC and if present, specific inhibitors of these targets may be useful in tumor therapy. For example,

several studies have shown that Cox-2 inhibitors in dogs may have antitumor effects in tumors

expressing Cox-2 [68, 69]. Also ICC can be used in diagnostic procedures and even allow identification

of markers for targeted therapies [28]. For example the use of ICC for targeted therapies was proved

with the analysis of estrogen and progesterone receptors in human patients with inoperable or

metastatic breast cancer [28, 70, 71]. Also in the workup of a thyroid nodule in human medicine, FNAs

are considered as a reliable, non-invasive diagnostic procedure for primary triaging of the patient. A

study reported that it was possible to detect a mutation of v-Raf murine sarcoma viral oncogene

homolog B1 (BRAF) by ICC on cytologic smears. This mutations are specific for papillary thyroid

carcinoma’s in humans and may confer a worse prognosis [72].

16

CHAPTER 3. Molecular markers in canine thyroid tumors

3.1 Molecular markers for prognosis

Differentiating follicular and medullary thyroid tumors

As mentioned before, it is difficult to distinguish dFTC and MTC by microscopic evaluation alone.

Therefore, immunohistochemical evaluation of histologic slides is required for this differentiation [24]

(Figure 4). Thyroglobulin, produced by the follicular cells of the thyroid, and calcitonin, produced by the

C-cells are two commonly used immunohistochemical markers. Different studies show that thyroglobulin

is detected in 90-100% of canine follicular carcinomas and calcitonin is detected in 70-100% of

medullary carcinomas [73]. The distinction between these two types of thyroid carcinoma might be

important concerning prognosis. In humans, MTC are shown to be biological malignant more often than

dFTC [74]. A study in dogs performed by Carver et al. (1995) determined that MTC were more likely to

be well circumscribed and resectable and might possess a less malignant nature than FTC [24].

However a recent retrospective study by Campos et al. (2014) concluded that the presence of distant

metastasis at time of diagnosis was not significantly different between canine patients with dFTC and

MTC. The investigators also looked at overall survival, disease-free interval, time to distant metastases

and time to loco-regional recurrence after thyroidectomy. Also in these parameters, no difference was

seen between dFTC and MTC. However, they found that MTC were less likely to be locally invasive at

presentation and hence more amenable to complete surgical resection, although outcome after

thyroidectomy was comparable between dFTC and MTC [3].

Cellular proliferation markers

Proliferation activity of a tumor, meaning the fraction of cells in the S-phase of the cell cycle, has been

shown to be predictive of the tumor’s biological behavior, for example growth rate and manifestation of

metastasis. Consequently, high proliferation activity is found to be associated with an adverse prognosis

[75, 76]. The detection of cell-cycle specific antigens such as proliferating cell nuclear antigen (PCNA)

and Ki-67 in neoplastic cells by IHC is therefore an important technique to assess prognosis in different

malignancies [77]. PCNA is a subunit of DNA polymerase-delta and essential for both replication of DNA

and repair of DNA errors. It has maximal expression in the G1 and S phase of the cell cycle [78, 79].

Ki-67 on the other hand is a large protein that is present in all phases of the cell cycle, except G0 [80].

Immunohistochemical labelling of Ki-67 (Figure 4) and PCNA are both reported to be related to

prognostic features in different types of tumors in dogs [77, 81-83]. Especially Ki-67 is widely

investigated in several canine tumors as an indication for prognosis. For example in canine cutaneous

mast cell tumors, a high Ki67 expression was associated with increased mortality, higher rate of local

recurrence and metastasis [84-86]. Furthermore, also in canine perineal gland neoplasms, Ki-67 is

effective in helping classification and to refine diagnosis criteria. Also in this tumor type, higher Ki-67 is

related to local recurrence of the tumor [87]. Correlation between high Ki-67 labelling index and

metastasis, death from neoplasia, low disease-free survival rates and low overall survival rates was also

seen in canine mammary tumors, showing the prognostic value of Ki-67 also in this type of tumor [81].

The prognostic value of Ki-67 was also tested on cytologic specimens of canine mammary tumors, which

17

revealed that immunocytochemical detection of Ki-67 in cytologic specimens correlated with that of

histologic specimens [88]. In human thyroid carcinomas, the value of Ki-67 as a prognostic marker was

investigated in several studies [89-92]. Both FTC and MTC that had metastasized had higher Ki-67

indices than tumors without metastasis [89, 91]. Also, in medullary thyroid carcinomas, results show that

the higher the Ki-67 index, the shorter the survival. In a study on IHC expression of Ki-67 in canine

thyroid carcinomas, Ki-67 index at time of diagnosis was positively associated with local invasiveness,

but not with distant metastasis [3].

Figure 4: Immunohistochemical expression of molecular prognostic factors in canine medullary thyroid carcinoma

A: Staining for calcitonin in a canine medullary thyroid carcinoma (400x). IHC evaluation of histologic slides by staining for

calcitonin, produced by thyroid C-cells, is important for the differentiation between dFTC and MTC. B: Staining for Ki-67 in a canine

medullary thyroid carcinoma (labeling index 28,4%). High Ki-67 expression levels are associated with increased mortality, higher

rate of local recurrence and metastasis [84-86]. Therefore, IHC analysis of Ki-67 expression in canine thyroid carcinomas could

serve as an indicator of prognosis.

(From: Campos, M., et al., Clinical, pathologic, and immunohistochemical prognostic factors in dogs with thyroid carcinoma. [3])

3.2 Molecular markers as potential therapeutic targets

Cyclooxygenase-2

Cyclooxygenase-2 (COX-2) is an inducible enzyme involved in the synthesis of prostaglandins out of

arachidonic acid [93] and is thus a key modulator of inflammation [94]. The first time the role of COX-2

in oncogenesis was suspected, was after epidemiological studies that revealed that the regular intake

of low doses of aspirin reduced the risk of colorectal cancer [95]. Later, studies revealed that COX-2

was overexpressed in colorectal cancers [96] and since then, upregulation of COX-2 expression has

been investigated in different types of human tumors. COX-2 is upregulated in the early phases of

oncogenesis [97] and in several human neoplasms this upregulation of COX-2 expression has already

been examined [98]. Molecular pathology studies have revealed that COX-2 is overexpressed in cancer

and stroma cells during tumor progression [93, 99].

18

In humans, epithelial neoplasms are very likely to have upregulated COX-2 expression. For example

colorectal cancer [97, 100], gastric carcinoma [101], bladder carcinoma [102], mammary carcinoma

[103] and also thyroid carcinoma [104] are proven to have high levels of COX-2 expression. Studies on

the biological effects of this upregulation have revealed that COX-2 promotes malignancy [94], increases

angiogenesis [105, 106], is correlated with metastasis [105, 106] and causes impairment of the immune

system by influencing regulatory T cell function and affecting activity of cells with cytotoxic function

[94, 98, 107, 108]. This leads to an increased survival of malignant cells and makes them resistant to

apoptosis [93, 94]. Also in human thyroid carcinoma expression of COX-2 and VEGF is thought to

promote angiogenesis, infiltration and metastasis [109]. Therefore, nonsteroidal anti-inflammatory drugs

(NSAIDs) and COX-2 selective inhibitors have promising therapeutic potential in cancer treatment.

Different studies have shown that COX-2 inhibition can attenuate tumor growth, decrease expression of

cell proliferation markers and promote apoptosis in tumor cells, even by COX-2 independent

mechanisms [110].

Accordingly, different canine tumors overexpress COX-2 as well; for example mammary tumors,

prostatic carcinoma, transitional cell carcinoma of the bladder and squamous cell carcinoma [111].

Likewise, different clinical studies on the use of NSAIDs and specific COX-2 inhibitors in canine

neoplasms were carried out. For example, in a study including 35 dogs diagnosed with prostatic

carcinoma, survival time in dogs that were treated with piroxicam and carprofen (non-specific COX-2

inhibitors) was significantly higher than in dogs that were not treated with NSAIDs [112]. Also in canine

transitional cell carcinomas of the bladder, treatment with NSAIDs showed good results. Complete and

partial remission were observed in some cases, as well as stabilization of the tumor in other dogs. The

response was associated with the induction of apoptosis and a reduction of a proangiogenic factor in

the urine (bFGF) [113]. However, in none of these two studies expression of COX-2 was useful to predict

response to treatment with NSAIDs, concluding that determination of the levels of COX-2 in a tumor

does not appear to be a good prognostic factor, nor a good indicator for the response to NSAID therapy

[111].

Beside tumor suppression by both COX-2 dependent and COX-2 independent mechanisms, COX-2

inhibitors can also produce synergic effects in combination with other anti-cancer therapies [114]. For

example COX-2 inhibitors, such as celecoxib, can have synergistic effects with radiotherapy in killing

malignant cells [115]. They improve the radio-sensitivity of tumoral cells by several mechanisms

including G2/M phase arrest [116]. Furthermore celecoxib is proven to decrease adverse consequences

of radiation therapy on normal cells [117]. Also in tumors (for example lung tumors) resistant to

chemotherapy due to the overexpression of P-glycoprotein (P-gp), celecoxib is effective in counteracting

this overexpression, making the tumor cells sensitive to chemotherapy. This effect is mainly obtained

through non-COX-2 pathways [118]. Also in vitro studies in medullary thyroid carcinoma cells have

proven that COX-2 inhibitors can reverse multi-drug resistance by inhibiting the expression of P-gp [119].

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) and its receptor (vascular endothelial growth factor

receptor 2, VEGFR-2) are the key regulators of angiogenesis [120, 121]. New blood vessel formation is

19

a physiological process used by many cancers to enhance their growth by providing oxygen and

nutrients and it enables metastasis as well [120, 122, 123]. Indeed, it is stated that tumoral masses need

new blood vessels in order to grow bigger than a few millimeters, hence they produce various

proangiogenic factors such as VEGF to stimulate mitosis of endothelial cells [124, 125].

Therefore, targeting the VEGF/VEGFR-pathway has been subject of new antitumor strategies in the

past decades [122]. A well-known substance used in anti-VEGF therapy is Bevacizumab, a humanized

murine monoclonal antibody against VEGF-A [126] which prohibits interaction between VEGF-A and its

receptors by binding circulating VEGF-A [127]. By suppressing the VEGF/VEGFR-pathway, growth of

neoplastic vessels is impaired which contributes to tumor growth restriction [121]. Indeed, clinical trials

showed promising results: the therapy effectively reduced blood vessel formation, tumor size and

metastasis [121, 128]. In practice this could mean increased progression free survival as well as better

overall patient survival rates and opportunities for new combinatorial therapies [128]. Likewise, several

VEGF targeted inhibitors have shown to improve the prognosis of human patients compared to

chemotherapy alone [120]. For example, in patients with metastatic renal cell carcinoma, bevacizumab

increased time to disease progression [129] and when combined with other chemotherapeutic drugs, it

improved prognosis in patients with metastatic colorectal cancer [130], breast cancer [131] and lung

cancer [127, 132].

P-glycoprotein

A frequently encountered problem in antitumoral treatments with chemotherapeutic drugs is the

so-called multidrug resistance (MDR) phenotype of tumoral cells, in which there is an overexpression of

the plasma membrane drug efflux pump P-glycoprotein (P-gp) [133]. P-gp is able to bind, transport and

remove chemotherapeutic agents out of the malign cells in an ATP-dependent manner [134], which

leads to resistance of the tumoral cells towards a broad range of pharmacologically unrelated

chemotherapeutic drugs, including vinblastine, vincristine and doxorubicin [135, 136]. Expression of

P-gp in cancer cells can be either intrinsically, because of the nature of the originating cells or acquired,

by selection or adaptation of cells during exposure to anticancer drugs [137].

The mechanism of P-gp mediated resistance is found to be more complex then first considered. Indeed,

besides the drug efflux activity of P-gp, also unintended indirect effects of P-gp have been described;

for example the influence of P-gp on the function of proteins involved in regulatory pathways such as

apoptosis [137]. P-gp-mediated MDR frequently means a restraint in effective chemotherapeutic

treatment of patients and therefore P-gp is considered to be an applicable molecular target in antitumoral

treatment [137]. Human MTC are refractory to conventional chemotherapeutic treatment in around

80-90% of the cases [138]. It has been shown that MDR plays an important role in this

chemo-resistant phenotype, which is reversible by targeting P-gp [139]. Campos et al. [4] studied the

expression of P-gp in canine MTC and showed the presence of P-gp in 70% of the investigated tumors,

making it a possible molecular target for treatment. Hence, anti-P-gp treatment could make canine MTC

more sensitive to chemotherapy and improve outcome of treatment [4].

20

Figure 5: Immunohistochemical expression of molecular therapeutic targets in canine thyroid carcinoma

A: Immunohistochemical expression of Cox-2 in a FTC of follicular-compact type with labeling index of 6.8% (400x).

B: Immunohistochemical expression of Cox-2 in a MTC with a labeling index of 22.4% (400×). C: Immunohistochemical expression

of VEGF in a FTC of compact type with 76-100% of positive neoplastic cells (400×). D: Immunohistochemical expression of VEGF

in a MTC with 76-100% of positive neoplastic cells (400×). E: Immunohistochemical expression of P-gp in a FTC of compact type

(400×). F: Immunohistochemical expression of P-gp (C494) in a MTC (400×).

(From: Campos, M., et al., Immunohistochemical expression of potential therapeutic targets in canine thyroid carcinoma. [4])

21

CHAPTER 4. Aims

This Master’s Dissertation builds further the research done by Campos et al. which revealed that

molecular markers could be useful in canine thyroid tumors to indicate prognosis and possibly also as

therapeutic targets. Given the fact that biopsies needed to investigate the presence of this molecular

markers are often unavailable, we wanted to validate immunocytochemical analysis of these markers

on FNA samples from thyroid carcinomas. The aim of this Master’s Dissertation was to standardize the

preparing and ICC staining of cytological smears of thyroid carcinomas after FNA collection and to

correlate ICC findings to the validated IHC techniques by Campos et al. [3, 4].

First, we aimed to investigate the possibility to distinguish canine FTC from canine MTC based on the

ICC expression of calcitonin and thyroglobulin. Second, we aimed to evaluate the immunocytochemical

expression of the therapeutic molecular markers Cox-2, VEGF and P-gp.

Even though both techniques are based on detection of molecular markers by antibodies, extrapolation

from validated IHC techniques to ICC is complex, given the considerable differences between the nature

of the specimens. Where IHC is performed on paraffin-embedded formalin-fixed sections or on frozen

tissue sections, ICC is carried out on cytological smears where tissue architecture has disappeared and

only individual cells are examined [12]. These differences result in technical challenges when

effectuating staining of cytological specimens with ICC. In this Master’s Dissertation, we aimed to

overcome these technical particularities and validate ICC in canine thyroid tumors on FNA samples.

To do so, we aimed to evaluate the influence of fixation techniques, storage temperature and storage

time to analysis on staining outcomes. To reach this goal, we applied different preparation protocols to

cytological smears of lymphoid tissue (Lymphonodus Popliteus) and evaluated the effects on CD3

staining outcome. Afterwards, we aimed to apply the protocol producing most consistent results to

positive control samples and eventually to cytological smears of thyroid carcinoma tissue.

22

CHAPTER 5. Material and methods

5.1 Case selection and selection of control samples

Canine patients diagnosed with thyroid carcinoma and treated with thyroidectomy at the Ghent

University Small Animal Department between May 2016 and December 2016 were included (n=7). In

each case histopathological confirmation of the diagnosis was performed as well as

immunohistochemical analysis of the tissue.

Control samples of thyroid, liver, kidney and lymph nodes were obtained from deceased canine patients

at the Ghent University Small Animal Department.

5.2 Cytological specimens and sample preparation

Thyroid carcinomas

Immediately after surgical excision of the thyroid mass, samples were taken. FNA samples were

obtained from the thyroid tumor using a 19G needle directed in different angels. Cells were brought onto

Histobond+ slides using a 5 CC syringe and smeared out over the slide, where after cells were

immediately fixed in ice cold methanol for 5 minutes. Then, the thyroid mass was cut in the middle and

impression smears were obtained by stamping the mass onto 10 Histobond+ slides after removing

redundant blood with absorbing paper. All slides were carefully identified with patients number, name

and type of sample. Finally, the tumor was fixed in formalin for histopathological and

immunohistochemical analysis.

Control samples

Positive control samples were used to evaluate the outcome of the used preparation and staining

protocols. Thyroid samples were used as positive control for staining with thyroglobulin, calcitonin and

VEGF, whereas smears of liver tissue served as positive controls for P-gp staining, smears of kidney

tissue for COX-2 and smears of lymphoid tissue obtained from the Lymphonodus Popliteus for CD3.

Control samples of thyroid, liver and kidney tissue were obtained from deceased canine patients as

soon as possible and at the latest 12 hours after death, using a 19G needle directed in different angels.

Cells were brought onto Histobond+ slides using a 5 CC syringe and smeared out over the slide,

afterwards cells were immediately fixed in ice cold methanol for 5 minutes. Thereafter, slides were stored

at room temperature and analyzed between 3 and 8 days after sample preparation.

Control samples of lymphoid tissue were obtained by puncturing the Lymphonodus Popliteus of recently

deceased canine patients, using a 19G needle directed in different angels. Cells were brought onto

Histobond+ slides using a 5 CC syringe and smeared out over the slide. In this manner, 9 cytologic

slides were prepared from every animal. Except for Animal 2, where one slide fell to the floor, so only 8

slides were used for analysis and Animal 3, where one extra slide was used to compensate the loss of

the slide of Animal 2. The table (Table 2) showed below shows the fixation and storage modalities of

the slides.

23

Animal Fixation technique Storage temperature Time of ICC analysis

Animal number 1 Methanol 3 slides RT: 1 slide Day 1

-20°C: 1 slide Day 1


Acetone 3 slides RT: 1 slide Day 1



Formalin 3 slides RT: 1 slide Day 1











-80°C: / slide Day 5



















Table 2. Overview of used fixation methods, storing temperature and storage time on lymphoid tissue taken from the

Lymphonodus Popliteus before ICC staining, in order to review the effects of sample preparation on ICC staining.

(RT: Room temperature)

24

5.3 Immunocytological analysis

Thyroglobulin

After incubating the fixed smears with 3% hydrogen peroxide in methanol for 5 minutes to quench

endogenous peroxidase, slides were washed twice with distillated water followed by two washes with

PBS (phosphate buffered saline). Then, slides were incubated with 100 µl of 30% goat serum for 30

minutes by 25°C. Thereafter, slides were washed twice with PBS and incubated with 100µl of the primary

antibody (polyclonal rabbit anti-human thyroglobulin, A0251, by Dako, Glostrup, Denmark) at a 1/6400

dilution for 2 hours by 25°C. 100 µl of the secondary antibody, biotinylated goat-anti-rabbit (Dako), at a

1/500 dilution was added after 2 washes with PBS. After incubation for 30 minutes by 21°C, slides were

washed twice with PBS and 100 µl of Avidin-Biotin Complex (ABC) with Horseradish Peroxidase (HRP)

was added to the slides and incubated for 30 minutes by 21°C. Sides were washed twice with PBS and

incubated with 500-700 µl of Diaminobenzidine solution (DAB) for 5 minutes at room temperature.

Afterwards, cells were counterstained with hematoxylin and mounted with aquatex.

Calcitonin

After incubating the fixed smears with 3% hydrogen peroxide (Dako kit Ref.K4011) for 5 minutes to

quench endogenous peroxidase, slides were washed once with distillated water followed by a wash with

PBS. Then, slides were incubated for 1 hour at room temperature with the primary antibody (polyclonal

rabbit anti-human calcitonin (Dako, Ref.A0576) at a 1/400 dilution in primary antibody diluent with

background reducing components (Dako, Ref.S302283). Afterwards, slides were washed with PBS and

incubated for 30 minutes at room temperature with the secondary antibody, Envision Link anti-rabbit

(Dako, kit Ref.K4011). Slides were washed twice with PBS and incubated for 5 minutes with DAB

solution (Dako, kit Ref.K4011) at room temperature. Afterwards, slides were washed with distillated

water, counterstained with hematoxylin and mounted with aquatex.

COX-2



PBS. Then, slides were incubated for 30 minutes at room temperature with the primary antibody

(polyclonal goat anti-Cox-2 (M19), Santa Cruz Biotechnology, Ref.sc-1747, Tebu-Bio) at a 1/400 dilution

in primary antibody diluent with background reducing components (Dako, Ref.S302283). Afterwards,

slides were washed with PBS and incubated for 15 minutes with the secondary antibody, rabbit anti-

goat (Dako, Ref.E0466), at a 1/400 dilution. After that, slides were again washed with PBS and

incubated with Envision Link anti-rabbit (Dako, kit Ref.K4011) for 30 minutes. Slides were washed twice

with PBS and incubated for 5 minutes with DAB solution (Dako, kit Ref.K4011) at room temperature.

Afterwards, slides were washed with distillated water, counterstained with hematoxylin and mounted

with aquatex.

25

VEGF



PBS. Then, slides were incubated for 1 hour at room temperature with the primary antibody (monoclonal

mouse anti-VEGF (SPM225), Santa Cruz Biotechnology, Ref.sc-65617, Tebu-Bio) at a 1/25 dilution in

primary antibody diluent with background reducing components (Dako, Ref.S302283). Afterwards,

slides were washed with PBS and incubated for 30 minutes at room temperature with the secondary

antibody, Envision Link anti-mouse (Dako, kit Ref.K4007). Slides were washed twice with PBS and

incubated for 5 minutes with DAB solution (Dako, kit Ref.K4007) at room temperature. Afterwards, slides

were washed with distillated water, counterstained with hematoxylin and mounted with aquatex.

CD-3



Dako Washing Buffer (Dako, kit Ref.K4011) . Then, slides were incubated for 30 minutes at room

temperature with the primary antibody (Dako CD3, Ref. A0452) at a 1/100 dilution in primary antibody

diluent with background reducing components (Dako, Ref.S302283). Afterwards, slides were washed

again with the Dako washing buffer and incubated for 30 minutes at room temperature with the

secondary antibody, Envision Link Labelled Polymer-HRP anti-rabbit (Dako, kit Ref.K4011). Slides were

washed twice with the Dako washing buffer and incubated for 5 minutes with DAB solution (Dako, kit

Ref.K4011) at room temperature. Afterwards, slides were washed with distillated water, counterstained

with hematoxylin and mounted with aquatex.

All slides were stained automatically by the Dako Autostainer+.

26

CHAPTER 6. Results

6.1 Thyroid tumor molecular markers

After fixation of the cytological smears, we attempted to apply the known techniques for IHC, as

described by Campos et al. [3, 4] on the cytological smears of our positive control samples (thyroid, liver

and kidney tissue). First of all we applied the classical staining protocol for IHC on the FNA samples. In

this way, one thyroid slide was stained for thyroglobulin and another one for calcitonin. Two slides with

cytological smears of liver tissue were colored for P-gp and two slides with cytological smears of kidney

tissue were colored for COX-2. Though, none of this colorations showed good staining quality on the

slides; the cellularity of the slides was low and much background coloration was present.

Given the major technical differences between IHC and ICC, this negative results were not unexpected.

An important issue to optimize IHC staining, is to use appropriate antibody dilutions for staining. It is

important to apply the highest possible antibody dilution to obtain a good staining signal without

background staining [140]. Therefore we opted to set up antibody dilution series for P-gp (on liver tissue

smears), COX-2 (on kidney tissue smears), VEGF and calcitonin (on thyroid tissue smears). However,

neither of these dilution series could enhance staining outcomes. Furthermore, IHC, which is performed

on paraffin-embedded formalin-fixed sections, requires antigen retrieval techniques prior to staining

because formalin fixation inactivates antibody binding sites in the tissue [141]. In general, acetone

fixation does not require antigen retrieval, but nevertheless we wanted to excluded that inactive antibody

binding sites were the reason for the negative results we got thus far. As we expected, also adding an

antigen retrieval step in the COX-2 staining protocol of kidney smears was not able to improve staining

outcome.

6.2 Lymphoid markers

The reason for the repeatedly negative staining outcomes on positive control samples remained thus

unclear. A lot of parameters in the preparation of the samples can influence staining outcomes in ICC

of cytological smears. For example fixation technique, storage temperature and storage time to analysis

can be of great importance to obtain good staining results. Because of the delicate nature of cytological

smears regarding ICC staining, it is often unclear which step in the preparation process is negatively

influencing coloration.

Therefore, we opted to evaluate different sample preparation protocols in order to identify optimal

sample preparation conditions. We decided to evaluate these different parameters (fixation technique,

storage temperature and storage time to analysis) on cytological smears of lymphoid tissues stained

with CD3 antibody, because it is proven to be a reliable antibody with good staining outcomes, also in

unfavorable circumstances [12-14]. We aimed to elect the preparation protocols giving the highest

reproducible results on lymphoid tissue and apply these protocols to thyroid tissue in order to validate

ICC staining of molecular therapeutic markers in thyroid carcinoma on FNA smears.

27

After staining, we noted that the majority off the cells in the slides were lipocytes instead of lymphoid

cells, which made it challenging to evaluate the influence of the different preparation protocols regarding

CD3 antigenicity maintenance. However, we were able to demonstrate that slides fixed in acetone

contained certainly less cells than slides fixed with methanol or formalin (Figure 6). This illustrates, as

described before [12], that cells fixed in acetone are less attached to the slide and therefore are more

easily washed away during the automated staining process. Further, we evaluated the lymphoid cells

present on the slides and we perceived that CD3 staining outcome was best in acetone fixed slides.

Though this is inconsistent with earlier published results, stating that formalin fixation gives best staining

results in CD3 staining [15]. Regarding storage temperature, best results were obtained in slides

preserved at a temperature of -20°C (Figure 7). Although we first intended to investigate the effect of

storage time to analysis on staining outcome by staining different slides that underwent the same

preparation protocol after 1 day, 1 week and 1 month, eventually we analyzed all slides within 5 days

after fixation. This was done because sample quality of first analyzed slides was poor and we concluded

that it would be complicated to review different protocols. Therefore we could not assess the effect of

storage temperature and type of fixation on durability of antigenicity over time.

Figure 6: Cell adhesion to the slides depends on fixation technique

Sample fixation protocols influence largely the outcome of ICC staining of slides. Regarding cell adhesion to the slides, cells fixed

in acetone are less attached to the slide then cells fixed in formalin or methanol, which leads to an important loss of cells during

the staining process. A: staining after acetone fixation after storing at -80°C (20x). B: staining after formalin fixation after storing

at -80°C (20x). C: staining after methanol fixation after storing at -80°C (20x). D: staining after methanol fixation after storing at

room temperature (20x).

28

Figure 7: Staining outcomes after ICC staining of lymphoid cells for CD3 after different preparation protocols

We evaluated differences in staining outcome after ICC staining of lymphoid cells for CD3, after different preparation protocols.

We noted that staining was best in acetone fixed slides. Moreover, freezing of slides after analysis improved staining outcomes.

Best staining results were seen after acetone fixation by a storage temperature of -20°C. A: staining for CD3 on a slide fixed in

acetone and stored by -20°C (20x). B: staining for CD3 on a slide fixed in acetone and stored at room temperature (20x).

C: staining for CD3 on a slide fixed in formalin and stored by -20°C (20x). D: staining for CD3 on a slide fixed in methanol and

stored by -20°C (20x).

29

Discussion

Thyroid tumors represent 1.2 to 3.8% of all canine tumors and are therefore the most common form of

endocrine neoplasia in dogs [1, 2]. They are mostly non-productive and their biological nature is

generally malignant [1, 29], resulting in high rates of metastatic disease observed at time of diagnosis

[2]. Hence, detection of prognostic and therapeutic molecular markers on thyroid FNA samples of each

patient could mean an easy and powerful method to enhance treatment outcome.

Sample preparation protocols

Control samples as well as thyroid carcinoma samples were taken and fixed as quick as possible after

death (< 12 hours) or immediately after tumor excision respectively. This is important for IHC and ICC

analysis, because cellular decay occurs fast and influences the detection of molecular markers. After

methanol fixation, slides were stored at room temperature for several days before analysis. Although

storage at room temperature has been proven to be appropriate if slides are analyzed within 1-2 weeks,

it is recommended to conserve slides at -20°C or even at -70°C for later analysis. Stored at -70°C,

cytological smears can maintain antigenicity for over one year [12, 28]. It is thus possible that this

suboptimal storage conditions led to loss of antigenicity and therefore influenced our staining outcome,

resulting in only little amount of specific staining on the slides. Besides, disappointing staining outcomes

in control samples may also be due to the use of inappropriate positive control tissues (discussed below).

In addition, also much background staining was perceived, which could be due to the fact that direct

smears from FNA were used. Indeed it is known that FNA smears cause a lot of background staining

because of cytoplasmic fragments from ruptured cells, whereas cytospin preparations do not pose this

problem [28].

Furthermore it has been stated that most reproducible results can be achieved with acetone fixation,

nevertheless also methanol fixation, as we used, has been described [12, 28]. While executing different

fixation protocols, we noted that slides fixed with acetone tended to loose large amounts of cells during

the automated staining process, which was not seen in slides treated with methanol or formalin. These

findings are consistent with previously published literature [15]. Fixation in acetone may thus lead to

loss of cellularity and moreover also to loss of cytoplasm within cells, which makes it difficult to recognize

the specific cell type on stained slides [15]. These disadvantages could lead to a loss of diagnostic

power after acetone fixation. Formalin fixation could be used to resolve these problems as it favors

adhesion of cells to the slide’s surface and results in better preservation of cell morphology, although in

this case an antigen retrieval step has to be added to the staining procedure to recover antigenicity.

However, the possibility to use formalin fixation depends on the type of antibody to be used, because

not all antibodies are compatible with formalin fixation [12]. Hence, the ideal fixation protocol depends

on the tissue and the targeted antigen.

30

Positive control samples

Positive control samples were used to evaluate the outcome of the protocols. Thyroid FNAs, taken from

recently deceased (< 12 hours) euthyroid patients, were used as positive controls for staining with

thyroglobulin, calcitonin and VEGF. These controls were chosen in order to make it possible to compare

results found by ICC to earlier published results by Campos et al., based on IHC [3, 4]. However,

calcitonin is only present in thyroidal C-cells, which only represent a small number of cells between

follicles. Therefore it is very unlikely to obtain a sufficient number of C-cells on smears after FNA sample

collection for positive control of calcitonin staining. This is also true for renal macula densa cells, which

we intended to use as positive control for COX-2 staining. Renal macula densa cells (and also medullary

interstitial cells) are positive for COX-2 [142], but represent only a small number of the total cells in the

kidney. When collecting renal cells by FNA, it is very unlikely that precisely the region with macula densa

cells is punctured, and therefore most smears after FNA sample collection will be negative for COX-2.

In contrast with calcitonin, which is physiologically only produced in the C-cells of the thyroid, COX-2

expression is present in almost all inflamed tissues and therefore FNA samples of inflamed tissue would

be a viable alternative as COX-2 positive control. Anyhow, attention must be payed in this case because

cellular decay and therefore loss of antigenicity is often present in inflamed tissues, whereas living cells

are needed to correctly perform ICC analysis.

Since initial staining outcomes were poor, we tried to evaluate the influence of various fixation and

storage protocols on immunocytochemical CD3 staining of lymphoid tissue, obtained by FNA samples

from the Lymphonodus Popliteus of recently deceased dogs (< 12 hours). As discussed in the next

alinea, correct interpretation of these results was almost unachievable because of poor sample quality.

However, samples fixed with acetone contained certainly the lowest amount of cells among all fixating

techniques, which is consistent with previous findings [15]. Though, our finding that lymphoid cells for

ICC CD3 staining were best fixed in acetone and preserved at a temperature of -20°C is inconsistent

with other reports that evidenced that formalin fixation gives best reproducible results in ICC CD3

staining [15]. This could be due to poor sample quality or suboptimal effectuation of the staining

protocols. Another remark that may be put forward in this regard, is that setting up a standard protocol

for fixation and storage of FNA samples before immunocytochemical analysis, is presumably impossible.

Different tissues require different protocols and protocols should be adapted to the antibodies that will

be used later on in the process. For example, some antigens remain undetectable by ICC after formalin

fixation, even when adequate antigen retrieval is performed [15]. Consequently, before research or

diagnosis based on immunocytochemical analysis of molecular markers can be carried out, it is

necessary to evaluate fixation and storage protocols for each antigen/antibody separately. This means

an extra step every time a new molecular marker is used, involving additional time and money. However

the value of a standardized procedure to evaluated presence of a certain molecular marker, for example

in cancer research or diagnosis, may be extensive.

31

Lastly, controlling sample quality at the time of sample collection by Diff-Quik staining is advantageous

and should be performed. Cellularity, cell type, cell condition and blood contamination can be assessed

and if necessary, samples can be retaken immediately in order to ensure good sample quality before

ICC analysis. During practical work in this Master’s Dissertation, we did not evaluate sample quality by

Diff-Quik staining, resulting in less conclusive results after ICC staining. For example, when evaluating

fixation and storage protocols for ICC detection of CD3 in lymphoid tissue, results after ICC showed few

lymphoid cells. Instead, the majority of the cells were lipocytes, which made proper evaluation of the

protocols impossible. This could have been avoided by controlling sample quality with Diff-Quik staining

before performing ICC.

Role of molecular markers in thyroid carcinoma management

Considering that good local tumor control significantly reduces the risk of metastatic disease [9], this

should always be looked at as a critical point in thyroid carcinoma treatment. However, this seems to be

a troublesome goal to achieve. Therefore it is important to confirm diagnosis as soon as possible and

to start a systemic treatment instantaneously.

In the future, anti-VEGF therapy and COX-2 inhibition might facilitate such an early systemic treatment,

possibly after immunohistochemical or immunocytochemical detection of these therapeutic markers

after biopsy or FNA respectively. Anti-angiogenic and anti-inflammatory therapies have demonstrated

to improve treatment outcome in different types of cancer, in human as well as in canine medicine.

However, previous studies have shown that nor the detection of VEGF nor of COX-2 expression levels,

were useful to predict tumor response to bevacizumab or NSAID therapy respectively [111, 143-145].

This could possibly mean that it will be challenging to predict outcome of therapy with bevacizumab or

piroxicam, only by investigating tumor expression of VEGF or COX-2 also in canine thyroid tumors,

however no study on this subject has been carried out yet. This ostensibly odd finding could be due to

a lack of biological activity of the detected molecules by IHC or Western blot. This is strongly suspected

in the case of COX-2, because a study identified a lack of correlation between COX-2 detection and

production of PGE2 [111, 146]. Moreover, NSAIDs can also interact with tumor cells in COX-2

independent mechanisms, for example by alternating gene expression [147], which could muddle the

direct relation between presence of the enzyme and the anti-tumoral effects of NSAIDs. Also,

anti-inflammatory drugs are mostly used in combination with other chemotherapeutic drugs or other

anti-tumoral treatments and therefore also synergistic effects may lead to more efficient therapy

outcomes then could be predicted based on COX-2 presence only.

P-gp inhibitors might be useful in the future to combat tumors that express multi drug resistance and

make chemotherapy more efficient in these cases. Given that up to 90% of human MTC are refractory

to conventional chemotherapeutic treatment [138] and the fact that P-gp expression in canine MTC is

present in 70% of the tumors [4], anti-MDR therapy could mean a major step forward in the

chemotherapeutic treatment of canine thyroid carcinomas. Also because results of chemotherapeutic

drugs, such as doxorubicin and cisplatin, are disappointing: response rates of 30-50% are noted, but

improved survival times could not yet be obtained [43, 44]. However, the toxicity of P-gp inhibitors is

32

currently the major obstacle that excludes the use of this type of drugs in patients. This toxicity is due to

their non-specific action and their non-selectivity, which make them act also on systems required for

elimination of P-gp substrates, leading to accumulation and toxicity of other anticancer drugs

administered at the same time [133]. Therefore, research for non-toxic alternatives, such as plant-based

P-gp inhibitors, is needed and ongoing [148].

Despite the obstacles encountered today, molecular targets may play an important role in future

treatment of various cancers, in human as well as in veterinary medicine. They possibly enable systemic

and targeted therapies, adjusted to the specific needs of every individual patient. This could lead to

more effective therapies with better local tumor control and less cases of metastatic disease, resulting

in augmented survival times. Nevertheless, extensive research in molecular, pharmaco(toxico)logical

and clinical fields will be needed to obtain this ambitious goal.

Future directions

To validate ICC on molecular markers in canine thyroid tumors, more research is needed. It will be

necessary to develop a preparation and staining protocol for each individual marker that will be

validated. Regarding fixation techniques, formalin offers advantages compared to acetone, but antibody

activity after formalin fixation will have to be evaluated for each marker. Therefore, good sample quality

is needed and this should be controlled by Diff-Quik staining of the samples immediately after collection.

Also appropriate positive control samples will be essential in order to compare different protocols. To

validate COX-2 staining, cytological smears of inflamed tissue can be used instead of kidney tissue.

Regarding calcitonin, it will be much harder to find positive control samples, given it is only produced by

thyroidal C-cells. VEGF and P-gp are often expressed in neoplastic tissues [124, 125, 137, 149, 150]

and therefore (malignant) neoplastic tissues may be useful as positive control for these molecular

markers. However, in these cases IHC control should be carried out in order to assure VEGF or P-gp

expression in the tissue used as positive control. For optimal staining results, samples should be stored

at -20 or -70°C instead of room temperature and be analysed as quick as possible after sample collection

in order to preserve antigenicity as much as possible.

In current medicine, IHC remains far more common used then ICC because of specific technical

difficulties observed when performing ICC, which we also encountered during practical work in this

Master’s Dissertation. However, ICC could be a promising technique because it offers a lot of useful

benefits in daily practice compared to IHC, such as less invasive sampling, quicker analysis and

pre-surgical diagnosis [12]. Hence, validation of molecular markers by ICC on FNA samples would open

doors towards improved management of inoperable canine thyroid tumors.

33

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