Comparison of Macrophages and Lymphocytes in Non

Mestrado Integrado em Medicina Veterinária
Ciências Veterinárias
Comparison of Macrophages and Lymphocytes in
Non-diseased Endometrium and Feline Endometrial
Adenocarcinomas
Miguel Augusto Tavares Pereira
Orientador:
Professora Doutora Maria dos Anjos Clemente Pires
Co-orientador:
Professor Doutor John F. Edwards
UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
VILA REAL, 2012
Mestrado Integrado em Medicina Veterinária
Ciências Veterinárias
Comparison of Macrophages and Lymphocytes in
Non-diseased Endometrium and Feline Endometrial
Adenocarcinomas
Miguel Augusto Tavares Pereira
Orientador:
Professora Doutora Maria dos Anjos Clemente Pires
Co-orientador:
Professor Doutor John F. Edwards
UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
VILA REAL, 2012
Dedicada à Dona Maria de Lourdes, uma grande
senhora e melhor avó do mundo.
“O caminho é longo e tem muitas encruzilhadas. Aí,
podemos parar e pensar um pouco, porque temos
opções a tomar. Algumas fecham-nos portas, mas
podem abrir novas. Outras não são bem o que
queremos, mas é o que precisamos. Outras não são as
que precisamos, mas as que queremos. No fim disto
tudo, temos de escolher! Porque se não escolheres, não
vives!”
Abstract
In the queen, the uterus is the most common site within genital tract for the occurrence of
tumors, though contributing to only 0.29% of all cancers diagnosed in these animals. Although
considered a rare tumor in cats, late studies showed that feline endometrial adenocarcinomas
(FEA) can to be more frequent than once thought. Aware of the importance of the immune
system, through a dynamic relation, in tumor immunoediting, this study aimed to assess the
infiltration of immune cells in FEA.
Ten samples of papillary serous FEA were used, along with ten samples each in follicular
and luteal stages of the oestrous cycle (controls). Indirect immunolabelling was performed using
antibodies against macrophages, T and B lymphocytes (MAC 387, Ab-Serotec®, 1:100; CD3,
Dako®, 1:50; CD79, Cell Marque®, 1:75 respectively).
Infiltration of immune cells was assessed in two different layers on non-diseased
endometrial stroma and in tumors in a total of 20 fields (at objective 40x), and also in the
myometrium around the tumors.
There were significant differences between layers of non-diseased endometrium, with
higher numbers of T lymphocytes on surface layer of follicular stage and of B lymphocytes on
deep layer of luteal stage. Only on T lymphocytes there were significantly higher counting
values on tumors than on non-diseased uterus. However, it was noticed a main significant
increase of the three cells types on tumors with pyometra. Also, in the tumor peripheral tissue
macrophages showed a significant increase when pyometra was present. The presence of
myometrium invasion didn´t showed significant variations.
This work showed an overview of immune cell infiltration on non-diseased endometrium
and in FEA and gave some suggestions on the importance of the immune system for this type of
tumor in these animals.
I
Resumo
Na gata, o útero é o órgão do trato genital onde ocorrem com maior frequência tumores,
embora contribua apenas com 0,29% para todos os tumores diagnosticados nesta espécie animal.
Apesar de ser considerado um tumor raro, vários estudos têm mostrado a possibilidade do
adenocarcinoma do endometrio felino (FEA) poder ser mais frequente do que se pensava
inicialmente. Conscientes da importância do sistema imunitário, através de uma relação
dinâmica, na imunoedição dos tumores, este estudo teve por objetivo a avaliação do infiltrado
inflamatório em FEA.
Foram utilizadas dez amostras de FEA do tipo morfológico papilar seroso juntamente
com 10 amostras de cada uma das principais fases do ciclo éstrico da gata - folicular e luteínica
como controlos. A imunomarcação foi realizada pela técnica indireta utilizando anticorpos antimacrófagos, anti-linfócitos T e anti-linfócitos B (MAC 387, Ab-Serotec®, 1:100; CD3, Dako®,
1:50; CD79, Cell Marque®, 1:75, respetivamente).
A presença destes tipos celulares foi avaliada em duas diferentes camadas nas amostras
de útero sem doença e no tumor, num total de 20 campos cada (com a objetiva de 40x). Além
disso, neste último grupo de amostras, contabilizou-se também o miométrio na periferia do
tumor.
Observaram-se diferenças significativas entre as duas camadas do endométrio, com
contagem superior na camada superficial da fase folicular para os linfócitos T e na camada
profunda da fase luteínica para os linfócitos B. Nos tumores verificou-se um aumento
significativo de linfócitos T, e no caso de piómetra concomitante, há um aumento dos três tipos
celulares. Por último, a quando da avaliação destes tipos celulares no tecido periférico ao tumor,
notaram-se aumentos significativos no que respeita aos macrófagos aquando da presença de
piómetra. A presença de invasão do miométrio não mostrou variações significativas.
Este trabalho permitiu ter uma ideia geral da distribuição destes tipos celulares estudados
no endométrio sem doença e nos FEA, permitiu-nos refletir sobre a importância do sistema
imunitário nestes tipos de tumores para esta espécie animal.
II
General Index
Abstract ............................................................................................................................................ I
Resumo ........................................................................................................................................... II
General Index ................................................................................................................................III
Figure Index .................................................................................................................................... V
Graphics Index ................................................................................................................................ V
Tables Index ................................................................................................................................... V
Attachments Index .......................................................................................................................... V
Abbreviations ............................................................................................................................... VI
Acknowledgments ....................................................................................................................... VII
Chapter 1 - Introduction ..................................................................................................................1
1.1
Background ......................................................................................................................1
1.2
Feline estrous cycle .........................................................................................................2
1.2.1
General concepts..........................................................................................................2
1.2.2
Feline cycle stages .......................................................................................................2
1.3
Feline endometrial adenocarcinomas ..............................................................................5
1.3.1
Incidence and epidemiology ........................................................................................6
1.3.2
Morphological features ................................................................................................6
1.3.3
Clinical signs and diagnosis ........................................................................................8
1.3.4
Treatment and prognosis ...........................................................................................10
1.4
Tumor-suppressor mechanisms .....................................................................................10
1.4.1
Intrinsic mechanisms .................................................................................................11
1.4.2
Extrinsic mechanisms ................................................................................................12
1.4.2.1
Immunoediting .................................................................................................12
1.4.2.1.1 Elimination phase ........................................................................................14
1.4.2.1.2 Equilibrium phase ........................................................................................15
1.4.2.1.3 Escape phase ................................................................................................15
1.4.2.2
Inflammation and cancer ..................................................................................15
1.4.2.2.1 Macrophages ................................................................................................16
1.4.2.2.2. Lymphocytes ...............................................................................................18
1.4.2.2.2.1. T lymphocytes ......................................................................................19
1.4.2.2.2.2. B lymphocytes .....................................................................................20
Chapter 2 - Objectives ...................................................................................................................21
Chapter 3 – Material and methods.................................................................................................22
3.1
Biological material ........................................................................................................22
3.2
Sample selection ............................................................................................................23
III
3.3
Immunohistochemistry analysis ....................................................................................23
3.4
Quantification ................................................................................................................24
3.5
Statistical Analysis ........................................................................................................26
Chapter 4 – Results ........................................................................................................................27
4.1.
Macrophages ..................................................................................................................27
4.2.
B lymphocytes ...............................................................................................................30
4.3.
T lymphocytes ...............................................................................................................33
Chapter 5 – Discussion ..................................................................................................................36
Chapter 6 - Final considerations ....................................................................................................42
Chapter 7 – References ..................................................................................................................43
Chapter 8 – Attachments ...............................................................................................................50
IV
Figure Index
Figure 1. Queen´s reproductive cycle ..............................................................................................3
Figure 2. In situ carcinoma ..............................................................................................................8
Figure 3. Papillary serous carcinoma ..............................................................................................8
Figure 4. Clear cells carcinoma .......................................................................................................8
Figure 5. The three phases of cancer immunoediting ....................................................................14
Figure 6. TAMs pro-tumoral functions. ........................................................................................18
Figure 7. Definition of layers on normal uterus ............................................................................25
Figure 8. Definition of layers on tumor .........................................................................................25
Figure 9. Macrophages’ immunohistochemistry results................................................................27
Figure 10. B lymphocytes’ immunohistochemistry results ...........................................................30
Figure 11. T lymphocytes’ immunohistochemistry results ...........................................................33
Graphics Index
Graphic 1. Macrophages results ....................................................................................................29
Graphic 2. B lymphocytes results ..................................................................................................32
Graphic 3. T lymphocytes results ..................................................................................................35
Tables Index
Table 1. Description of the used tumor cases ................................................................................22
Table 2. Used immunohistochemestry technic’s specifications ....................................................24
Table 3. Descriptive analysis of macrophages results ...................................................................28
Tabel 4. Descriptive analysis of B lymphocytes results ................................................................31
Table 5. Descriptive analysis of T lymphocytes results ................................................................34
Attachments Index
Attachment 1. Analysis of variance of macrophages results .........................................................50
Attachment 2. Post Hoc test to macrophages results .....................................................................50
Attachment 3. Analysis of variance of B lymphocytes results ......................................................51
Attachment 4. Post Hoc test to B lymphocytes results ..................................................................51
Attachment 5. Analysis of variance of T lymphocytes results ......................................................51
Attachment 6. Post Hoc test to T lymphocytes results ..................................................................51
Attachment 7. Analysis of variance of immune cells counting on peripheral tissue whether
pyometra is or not present .............................................................................................................51
Attachment 8. Analysis of variance of immune cells counting between tumor and peripheral
tissue considering pyometra ..........................................................................................................51
Attachment 9. Analysis of variance of immune cells counting on tumor mean and peripheral
tissue whether there is or not myometrium invasion .....................................................................51
Attachment 10. Analysis of variance of immune cells counting between tumor and peripheral
tissue tissue considering invasion ..................................................................................................51
V
Abbreviations

X

Min – Minimum

AAM – Alternatively activated

MMP – Matrix-metalloproteinase
macrophages

NK cells – Natural killer cells

ANOVA – Analysis of variance

NO – Nitrous oxide

APC – Antigen presenting cell

OVH – Ovariohysterectomy

BSA – Bovine serum albumine

PBS – Phosphate buffered saline

CAM – Classically activated

Periph – Peripheral
macrophages

SD – Standard deviation

CTL – Cytotoxic T lymphocytes

Supf – Superficial

CV – Coefficient of variation

TAM – Tumor associated macrophages

DAB – 3,3'-Diaminobenzidine

TCR – T cell receptor

DNA - Deoxyribonucleic acid

TCR – T cell receptor

FEA – Feline endometrial

TGF – Tumor growth factor
adenocarcinoma

Th – T helper lymphocytes

IS – Immune system

TIL – Tumor infiltrating lymphocytes

LH – Luteinizing hormone

TNF – Tumor necrosis factor

LinfB – B Lymphocytes

TNFR – Tumor necrosis factor receptor

LinfT – T lymphocytes

TRAIL – Tumor necrosis factor related

m – Months

Mac – Macrophages

Treg – T regulation lymphocytes

MALT – Mucosal associated lymphoid

U – Unknown
tissue

WHO – World Health Organization

Max – Maximum

y – Years

MHC – Major histocompatibility
–Mean
apoptosis-inducing ligand
complex
VI
Acknowledgments
O atingir de mais uma etapa apenas foi possível pela presença e apoio de várias pessoas.
Correndo o risco de, ao nomeá-las, me esquecer de alguém, a todos deixo, desde já o meu
sincero agradecimento.
Começando a individualização, tenho, obrigatoriamente de começar pelos meus pais.
Sem eles, sem o apoio e força que sempre me deram, sem as longas conversas envoltas em
conselhos e ensinamentos, em “pancadas” e carinho, em amor e apoio, nunca teria conseguido
formar-me como pessoa e profissional. A eles e ao Marco, o meu irmão e melhor amigo, que
mesmo na outra ponta do país, sempre me apoiou, aconselhou e deu força para me ajudar a
terminar esta etapa, o meu muito obrigado. Também à minha restante família, em especial à
minha avó Lourdes, pelo apoio e carinho.
À Professora Maria dos Anjos pela orientação, ensinamentos, amizade e conselhos.
Nunca conseguirei agradecer-lhe o suficiente pelo apoio que me deu nas horas desesperantes de
trabalho laboratorial, ou nas longas tardes de escrita de tese, assim como pelos projectos e
trabalhos paralelos que fomos desenvolvendo.
Agradeço também à TAMU, em especial ao Professor John F. Edwards, mas também aos
restantes professores, internos e pessoas que conheci em College Station por me terem recebido,
pela oportunidade que me deram de poder aprender mais, aperfeiçoar a minha técnica de
necrópsia e vivenciar uma experiência diferente de trabalho. Agradeço novamente ao Professor
Edwards pela co-orientação deste trabalho, pelos ensinamentos e conselhos. E a ele e à Dona
Nair por me receberem de braços abertos em casa deles, por me tratarem como um filho e pela
paciência, amizade, carinho, conselhos e oportunidades que me deram de conhecer mais do
Texas do que apenas College Station. A minha estadia, graças a eles, foi uma experiência
refrescante, que me permitiu ganhar mais confiança no meu trabalho, aprender a vários níveis e
conhecer um país diferente.
Também à Professora Rita Payan-Carreira pelo apoio e amizade, pela co-autoria em
vários trabalhos, pelo conhecimento transmitido e conselhos e pelo facto de se mostrar sempre
disponível para dar uma “mão” ou para por em uso a minha capacidade crítica. À dra. Ana Laura
Saraiva pela amizade, apoio e conselhos. A ambas, pela participação no projeto no qual este
trabalho está inserido, o meu muito obrigado. Ainda ao Professor Jorge Colaço, pela ajuda na
sempre complicada estatística.
VII
Agradeço ainda novamente à professora Rita e ao Victor pela ajuda que me deram na
imagem 1. Sem as trocas de conhecimento, definições e escolha de termos corretos e trabalho
fundamental na parte gráfica, não a conseguiria fazer.
Agradeço também à UTAD pela oportunidade que me deram de fazer este curso, a todo o
pessoal docente e não docente que, de uma forma ou de outra, me ensinaram, influenciaram e me
ajudaram a terminar este curso.
Tendo passado grande parte do meu estágio no LHAP-UTAD, tenho obviamente de
agradecer à Dona Lígia Bento pelo excelente trabalho de apoio técnico no processamento do
material, à Dona Ana Plácido e Dona Glória Milagres pelo apoio técnico, disponibilidade,
amizade, descontração e ensinamentos. Agradeço também às restantes professoras do laboratório
pelas oportunidades que me deram de aprender, pelas conversas mais descontraídos e pelos
conselhos que me foram dando ao longo do meu estágio. Também aos meus parceiros de
laboratório, à Marta, companheira de laboratório e de muitas outras andanças, e ainda à Daniela,
Nuno, Raquel, Sara, Filipa e aos restantes, pelo que me ensinaram, pela descontração, amizade e
por tornarem o dia-a-dia no laboratório mais fácil.
Também à Armanda pela ajuda na revisão do inglês e apoio na escrita da tese.
À minha família da residência, o Davide, Telmo e Tozé, por me terem recebido em Vila
Real nos meus primeiros tempos fora de casa. Aproveito para juntar a este grupo o Victor, o meu
colega de quarto que sobreviveu mais tempo. A estes quatro, agradeço, em primeiro lugar, por
estarem presentes e por me permitirem saber que, chegando à residência, iria chegar a casa, onde
não iria estar sozinho. Foi com eles, com o nosso Tratado de Kinshassa, com as nossas conversas
noturnas, com as nossas brincadeiras e parvoíces “residenciais”, que consegui, apesar dos
tropeções e cabeçadas na parede, manter-me firme em Vila Real, no meu curso e na minha vida.
A eles e às outras pessoas com quem partilhei a minha vida de residência, como o Simão, a Tita
e o Alexandre, o meu muito obrigado.
Também à minha família de veterinários, com muito carinho à Xica, Fiúza, Di, Xeco e
Lucy os meus companheiros de curso e de aulas, de bebedeiras e de conversas sérias, de parvoíce
e festa, pela amizade e conselhos, pelo carinho e paciência, por toda a minha vida académica,
pelos tempos de AEMV. Ainda neste grupo de veterinários malucos terei ainda de incluir muitas
mais pessoas. Em primeiro lugar a Martinha, Té e Renata. Mas também muitos mais. Correndo o
risco de me esquecer de alguns, aqui vai: a madrinha Diana e ao meu padrinho Bernardo pelo
espírito académico e conselhos que me incutiram, à Ângela, Marta, Tati, Xico, Badano, Gritos e
todos os meus praxadores, presidenta Raquel, Vanessa, Cristóvão, Hélio, Marco e todos os meus
VIII
companheiros finalistas e de Dominicana, à minha afilhada Karin, Vânia, Marta, Mia e Maria,
pelas longas conversas, pelas vezes que me deram na cabeça, pela amizade e carinho e por
confiarem, de vez em quando, nos meus conselhos, ao João, à restante dúzia e meia de afilhadas
de praxe, aos meus restantes caloiros e restante família de praxe. A todos eles agradeço pelas
noites na tenda, pelas jantaradas e por me acordarem nas aulas, pelos conselhos e conversas mais
animadas, enfim, por estas e muitas mais coisas que alguma vez vou esquecer e que me
permitiram viver uma longa e feliz vida académica.
Agradeço ainda à AEMV-UTAD, a todos os meus companheiros “associativistas” e
professores que colaboraram em diversos eventos e que ajudaram a torna-los um enorme
sucesso. Esta experiência associativa foi enorme na minha formação pessoal e profissional e
permitiu-me uma grande abertura de horizontes.
Ao Leo e Cátia pelas longas conversas ao telemóvel ou num qualquer café em Albergaria,
pela amizade e carinho. Também à Marisa pelas aventuras na Vila e na Bila.
Ao Ricardo, Carla, Alex e Alice pela amizade. À Maria, Caracoleta, Daniela, Loura e
todos os restantes elementos da Tribo da Luz, ao João, Cat e aos meus meninos do Jorac e aos
meus amigos dos escuteiros. Todos vocês foram importantíssimos na minha vida e formação, nos
momentos em que pude esfriar a cabeça e sentir-me em casa em Valmaior.
Ao pessoal da Meo House e mais alguns de CM, a minha segunda casa neste último ano e
cuja companhia, momentos de relaxamento (alguns na altura errada), amizade e ajuda em
Photoshop sempre me safaram, à Sandrinha e às meninas, que sempre me mostraram com um
sorriso e uma palavra de carinho em todos os momentos. Também aos companheiros de Tuna e a
muitos mais com quem me cruzei nesta vida Transmontana.
Tendo noção que mais agradecimentos seriam necessários, mas que o espaço é limitado e
a memória também, agradeço a todos que estiveram na minha vida ao longo destes seis anos de
curso e vinte e muitos anos de vida, e peço desculpa aos de que me esqueci. Sem vocês, esta vida
não tinha piada nenhuma e este trabalho não seria possível!
IX
Chapter 1 - Introduction
1.1 Background
The cat species (Felis catus) has evolved from the African wild cat (Felis lybica) and has
become one of the most popular companion animals (Linnaeus, 1958; Goodrowe, 1992; Driscoll
et al., 2007). A considerable increase on the study of the feline reproduction has followed their
ever growing importance as pets (Chatdarong, 2003), moreover they have been used as a model
for research aimed at the preservation of wild felids (Wildt et al., 1986) as well as their use for
studying human physiologic abnormalities (Goodrowe et al., 1989)
Despite the uterus role in reproduction, pathologic changes occurring in the uterine tube
of dogs and cats have been reported infrequently (Gelberg & McEntee, 1986). In fact, uterine
tumors represent only 0.29% of all neoplasms diagnosed in cats (Miller et al., 2003). Being
considered extremely rare, uterine tumors in the cat include mesenchymal and epithelial origins
as fibroma, adenocarcinoma and a mixed mesodermal tumor (Papparella & Roperto, 1984).
Uterine adenocarcinoma are considered uncommon in domestic animals with exception for
bovines and rabbits (Preiser, 1964). Feline endometrial adenocarcinoma (FEA) is considered a
rare tumor; earlier studies only found a small number of cases (Miller et al., 2003; Gil da Costa
et al., 2009). In a 2012 study, a large number of cases were identified in portuguese cats and
concluded that the low number of previously identified cases could be due to many being
clinically silent and therefore undiagnosed. In fact, metastasis is the only clinical sign of FEA
easily detectable, as other clinical signs are nonspecific compared with those of pyometra
(Saraiva et al., 2012).
On the other hand, the importance of the immune system (IS) is increasingly taken into
account. In fact, the relationship between this system and the tumor is seen as a continuous
dynamic process during tumor genesis where, paradoxically, the IS acts both as an extrinsic
suppressor (through the immunosurveillance) and a promoter for tumor growth (inflammation as
a key point) (Vesely et al., 2011).
Therefore, taking into account the importance of the immune system in the biology of
tumors, we tried to characterize the immune response of feline endometrial tumors, comparing
the results obtained in tumors with the B and T lymphocyte and macrophage infiltrates in two
stages of oestrus cycle in apparently healthy uteri (devoid of lesions/disease). We sought to
contribute to the knowledge of the immune system’s response to these types of tumors.
1
1.2 Feline estrous cycle
Compared to the dog, there is still a lack of knowledge on the reproductive physiologic
mechanisms in the cat, leading to some doubts and controversy on some points (Fontbonne &
Garnier, 1998), such as nomenclature and characterization of cycle phases.
1.2.1 General concepts
Queens are classically described as seasonally polyestrous and induced ovulators, with
ovulation induced by coitus, usually more than once. However, in the absence of copulation,
spontaneous ovulation may occur in some queens, perhaps triggered by visual stimulation or
pheromone cues. Spontaneous ovulation occurs more frequently in young animals or when they
are found in large groups (Concannon et al., 1980; Mialot, 1984; Banks, 1986; Fontbonne &
Garnier, 1998; Johnston et al., 2001b; Little, 2001a).
The pubertal estrus occurs for most queens between four to twelve months of age, being
influenced by photoperiod and by the female body condition. Other factors affecting the onset of
puberty include the breed (long-haired females usually reach puberty later than short-haired
queens), social environment, health, physical condition and nutritional plan (Mialot, 1984;
Fontbonne & Garnier, 1998; Johnston et al., 2001b; Little, 2001a).
Having a positive response to the increase of day length (Cunningham, 2002; Brown,
2006), queens are long-day breeders, showing a prolonged anestrus during short-day length, that
might be represented by the period between September and December in the Northern
Hemisphere (Kutzler, 2007). The duration of anestrus may be shorter in southern regions.
However, the length of the anestrus season may be shortened if the female is kept indoors under
light and warm temperature (Johnston et al., 2001b; Brown, 2006). Meanwhile, queens start to
cycle in late January and February as days start getting longer. The presence of a queen in estrus
or a male is another factor that can provide stimulatory social stimuli for the onset of estrus
(Johnston et al., 2001b).
1.2.2 Feline cycle stages
As induced ovulators, queens can show two different sorts of estrous cycles whether
ovulation occurred or not.
They can show an anovulatory cycle, where, in the absence of ovulation no luteal stage
exists and recurrent follicular stages (proestrus and estrus) accompany the recurrent follicular
development waves (Mialot, 1984; Fontbonne & Garnier, 1998; Little, 2001a).
2
An ovulatory cycle is comprised of both follicular and luteal stages, the later having
different durations if pregnancy occurs, in the case of a fertile mating, or in the case of a nonpregnant diestrus, if the queen attains ovulation, either spontaneous or coitus-induced. The nonpregnant luteal stage can be prolonged by pseudopregnancy in the case of non-fertile copulation.
In the ovulatory cycles, the luteal stage can have different durations (Figure 1), that result in
different interestral intervals (Mialot, 1984; Fontbonne & Garnier, 1998; Johnston et al., 2001b;
Little, 2001b; Little, 2001a).
Figure 1. Queen´s reproductive cycle
After the period of seasonal anoestrus, the animal begins to cycle, having an anovulatory or ovulatory
cycle. After the various phases of the cycle the animal returns to proestrus and the next cycle of the
animal can keep or change their type, so in the same breeding season, the animal may have several
different types of cycle. Also, at the end of the reproductive season the animal returns to a seasonal
anestrus.
The interestrous period between one estrus and the next is often designated postestrus. In
this quiescent stage, plasma estradiol levels return to basal levels, and there is no sexual behavior
or receptivity. The interestral interval can refer to the period between two consecutive breeding
seasons (Banks, 1986; Stabenfeldt & Pedersen, 1991; Johnston et al., 2001b).
In the reproductive season, consecutive cycles occur, reflecting the major ovarian events.
As happens in some other domestic species, like the horse, the classical cycle stages are not
easily distinguished, and often two stages are tagged under a common name, such as follicular
3
stage (including proestrus and estrus) or luteal stage (including metaestrus and diestrus) (Banks,
1986).
In the ovulatory cycles classically consist of four stages: proestrus, estrus, metaestrus and
diestrus (Mialot, 1984).
Proestrus is a short stage (1-3 days long) with an irregular appearance in queens, as it
often cannot be distinguished, both in morphology and behavior, from estrus. Hence, some
authors refer that only a minority of females are observed in proestrus considering most are
observed in estrus directly after anestrus, postestrus or diestrus. At the hormonal level, it is
usually associated with the rise of serum estradiol concentrations, secreted by follicular
granulosa cells of developing follicles (Shille et al., 1979; Johnston et al., 2001b). As proestrus
is hardly distinguishable from estrus, the two stages are grouped together under the name of
follicular phase.
With the peak of follicular activity and estradiol secretion, estrus is the behavioral stage
of receptivity to mating. Its duration is highly variable, ranging from 2 to 16 days (an average of
7 days), depending on the animal. After the coital stimulus, an increased blood luteinizing
hormone (LH) concentration is found to occur, leading to subsequent ovulation with the
formation of a corpus luteum and the end of the follicular phase. The number of mattings will
determine the strength of the LH surge (both its length and its concentration) in triggering the
ovulation cascade, therefore determining the occurrence or not of the ovulation and the number
of ovulations. In this way, the LH surge indirectly controls both fertility and prolificity in the
species. Ovulation occurs 24-48 hours post-copulation, but this interval may be extended up to
90h in particular conditions (Shille et al., 1979; Concannon et al., 1980; Wildt et al., 1981; Shille
et al., 1983; Johnston et al., 2001b).
Luteal cells rapidly proliferate in ovulated follicles, and corpora lutea soon become
functional. In cats, it is not easy to distinguish the metaestrus from diestrus unless microscopical
evaluation of the ovaries and uterus is performed. These two stages are often called postestrus or
simply diestrus. Progesterone starts rising 24-48h after ovulation, closely following the
formation of corpora lutea. In non-pregnant cycles, the progesterone-dominated phase lasts
between approximately 30 days (diestrus) and 38-40 days (pseudopregnant queen), while the
length of pregnancy phase is of 60 days. Plasma progesterone concentrations remain similar for
the first 21 days in the pregnant and non-pregnant cycles, thereafter being lower in the nonpregnant diestrus (Paape et al., 1975; Verhage et al., 1976; Shille & Stabenfeldt, 1979; Wildt et
4
al., 1981; Banks & Stabenfeldt, 1983; Schmidt et al., 1983; Stabenfeldt & Pedersen, 1991;
Johnston et al., 2001b).
As previously mentioned, queens have a period of anestrus during the short-day season.
However, there is a short post-partum anestrus phase, that corresponds to the period of uterine
involution before resuming of estrous cycles. Uterine involution in cats is quite fast in
comparison to dogs, and in some cases, a queen can start estrus and become pregnant during late
lactation. During anestrus, plasma estradiol and progesterone concentrations are at basal levels
(Banks et al., 1983; Banks & Stabenfeldt, 1983; Johnston et al., 2001b).
1.3 Feline endometrial adenocarcinomas
The uterus is the most common site of tumors in the feline reproductive tract (Miller et
al., 2003). An uterine adenocarcinoma is a neoplasm consisting of malignant epithelial
endometrial cells (Kenedy et al., 1998), and in human medicine, endometrial cancer is the
seventh most common malignant disorder (Mandiæ, 2004; Amant et al., 2005; Wallace et al.,
2010), leading the scientific community to try to find animal models for study. This neoplasm
is considered rare in most domestic animals, except rabbits, cattle (Cotchin, 1964; McEntee &
Nielsen, 1976; Elsinghorst et al., 1984; Kenedy et al., 1998) and in virgin Han:Winstar rats
(Deerberg et al., 1981; Elsinghorst et al., 1984). The uterus is the most common site of tumors
in the feline reproductive tract.
The common practice of ovariohysterectomy in cats is thought to be protective from
uterine neoplasia (Miller et al., 2003; Taylor, 2010). However, there are reports of
adenocarcinoma in the incompletely removed uterine stump of neutered cats (Miller et al., 2003;
Anderson & Pratschke, 2011). Another explanation for the low prevalence of FEA in queens that
may be subjected to long periods of unopposed estrogen stimulation may be linked to the
practice of periodically breeding or inducing ovulation in queens (Stabenfeldt & Pedersen, 1991;
Cho et al., 2011). Furthermore, spaying of young queens for contraception is not a common
practice in purebred queens, nor is it a worldwide procedure. Saraiva and her collaborators
(2012), suggested that perhaps other reasons can explain the small number of FEA in cats like
the inadequate post mortem examination of the genital tract or the lack of interest in
anatomopathologic evaluation of ovariohysterectomy (OVH) surgical specimens.
5
1.3.1 Incidence and epidemiology
Endometrial adenocarcinoma is more common in the cat than in the dog and is usually
locally invasive (Morris & Dobson, 2001). However, reports of metastases are common and,
along with severe illness, they are considered responsible for the presentation of the animal at the
clinic (Cho et al., 2011).
While studying the epidemiology of a disease, breed can be an important factor. Previous
studies have demonstrated an increased incidence of FEA in purebred animals, although no
breed predisposition has been reported (Johnston et al., 2001a; Klein, 2007). This authors
proposed that this fact maybe was correlated with the longer reproductive life of purebred cats
relative to other domestic cats (Johnston et al., 2001a). However, Saraiva and collaborators
(2012), states that population ratios between intact purebred and mixed breed animals may
change with geographic location. They believe that, in Portugal, both purebred and mixed breed
queens all tend to be spayed late. In addition, among the uterine samples used in their study,
more cases of FEA were in crossbreed animals than in purebreds, reflecting the predominance of
the population of the former over the later (Saraiva et al., 2012).
Age is another important feature in the study of neoplasia study. A positive relationship
between increased age and the development of FEA has been found, with FEA being seen more
frequently in queens older than 9 years old (Preiser, 1964; Belter et al., 1968; McEntee &
Nielsen, 1976; Morris & Dobson, 2001; Klein, 2007). Yet, sporadic reports of FEA in animals
aged 5 or less are reported. Cho and collaborators (2011) and Saraiva and her colleagues (2012),
concerned in their studies animals ranged from 2 to 15 years, indicating that FEA can develop at
a younger stage.
1.3.2 Morphological features
Macroscopically, the epithelial tumors of feline uterus present as a diffuse thickening of
the endometrium along the uterine horns and corpus or as multiple, white nodules projecting into
the lumen. In the presence of pyometra, the uterine wall can be thinned. Furthermore, in invasive
tumors, tumor infiltration is found through the myometrium, being possible the production of an
protuberance-like lesion into serosa that, if ruptured, promotes neoplasic peritonitis(Saraiva et
al., 2012).
Albeit the lack of morphology description of these lesions in the current WHO
classification of tumors (Kenedy et al., 1998), Saraiva et al. (2012) recently classified FEA into
6
three different histologic types, based on their morphology and similar endometrial carcinoma
patterns in women: papillary serous carcinoma, clear cell carcinoma and “in situ” carcinoma .
Papillary serous carcinomas (Figure 2) were the most common type and have a papillary
growth into the lumen supported by a variable fibrovascular stroma lined by more than one layer
of neoplasic cells. The tumor is composed essentially of columnar shaped cells with a moderate
amount of eosinophilic cytoplasm that can have some clear spaces. Despite a predominant
papillary serous morphology, some areas can have solid growth, and others have a glomeruloid
pattern or a variable numbers of clear cells. The nuclei are round to oval with loss of polarity and
can be vesicular or hyperchromatic. Large nucleoli and sometimes intranuclear clear inclusions
can be observed together with other malignant features such as anisokaryosis, anisocytosis,
frequent bizarre mitotic figures and numerous multinucleated cells. Some areas of necrosis may
be found and, occasionally, psammoma bodies and calcification are noted. The tumor can invade
the myometrium, blood or lymphatics vessels, and in severe cases, the serosa may rupture.
Inflammatory foci with macrophages, plasma cells, lymphocytes, neutrophils and low numbers
of eosinophils can be observed. In some situations squamous metaplasia can also be found
(Belter et al., 1968; Saraiva et al., 2012).
Although less common, FEA can have other morphologies. The “in situ” carcinoma
(Figure 3) is morphologically very similar to the papillary serous carcinoma. The major
difference, as the name implies, is the lack of invasiveness with the tumor developing very
superficially in the apical endometrium and not even invading the submucosa (Saraiva et al.,
2012).
Clear cell carcinomas (Figure 4), the more infrequent type of FEA, are almost entirely
composed of clear cells arranged in papillae, sheets or solid nests surrounded by fibrovascular
stroma. Clear cells are large, round to polygonal with foamy cytoplasm and eccentric crenate or
ovoid nucleus with a prominent eosinophilic nucleolus. These types of tumors can also present a
moderate degree of anisokaryosis, anysocytosis and some foci of necrosis, as in the papillary
serous type, but inflammatory cells are rare and multinucleated cells are absent. In these types of
tumors, the invasion of the myometrium is not a constant feature and, despite the inexistence of
descriptions of neoplastic emboli in vessels, it possible occurrence cannot be discarded (Saraiva
et al., 2012).
7
Figure 3. Papillary serous carcinoma
Papillary
proliferation
of
endometrial
cells.
Hematoxilin and eosin stain, Bar = 100micra (with
LHAP permission)
Figure 2. In situ carcinoma
Neoplasic proliferation of endometrium on the surface
layer, with papilar proliferation. . Note the glands on
the deep endometrium are normal. Hematoxilin and
eosin stain, Bar = 100micra (with Saraiva et.al.
permission)
Figure 4. Clear cells carcinoma
Proliferation of the endometrium. Note the cell with
clarified cytoplasm. Bar = 30 micra (with Saraiva et.al.
permission)
1.3.3
Clinical signs and diagnosis
Most published studies of FEA include few details of its clinical aspects. Saraiva and
colleagues (2012) suggested that early stages of FEA probably evolve as a silent disease and
were only detected clinically in cases where large lesions, invasion, metastases or pyometra
existed. The clinical signs of FEA vary with the size of the lesion, the age of the process and the
presence and pattern of metastasis. The clinical signs seen may depend on the morphological
8
type of the tumor (Preiser, 1964; Belter et al., 1968; Anderson & Pratschke, 2011; Cho et al.,
2011; Saraiva et al., 2012).
The presence of a mass within the uterus, whose size may vary from less than one to ten
centimeters (Johnston et al., 2001a) in the absence of metastasis, may induce a chronic
inflammation (Taylor, 2010). Therefore, the result is that small-size masses are usually found
incidentally at ovariohysterectomy or post-mortem, while large masses produce varying severe
clinical signs (Taylor, 2010; Saraiva et al., 2012). By inducing inflammation within the uterus,
FEA may result in vaginal discharge that can be mucous, purulent or hemorrhagic, which can be
intermittent, or may be associated to pyometra (McEntee, 1990; Taylor, 2010). Major signs at
presentation can be similar to those of pyometra including: vaginal discharge, vomiting, irregular
appetite and polyuria/polydipsia, but these can be observed together with more localized signs
when associated with palpable enlargement of the uterus that causes abdominal distress and
distension (Johnston et al., 2001a; Taylor, 2010). A large tumor can compress adjacent viscera to
cause other signs, like constipation and dysuria (Belter et al., 1968; Klein, 2007; Saraiva et al.,
2012).
Illness is usually related to the presence of metastases (Saraiva et al., 2012) or
inflammation. In fact, as an indicative period, most cats develop disease within 6 months of
diagnose if a uterine excision is not performed or if there is already lymphatic invasion (Taylor,
2010). It must not be forgotten that most cases are diagnosed in later stages of the disease
(Saraiva et al., 2012). The primary signs associated to local or distant metastasis include ascites,
anorexia and weight loss, that can co-exist with regenerative anaemia or neutrophilia, fever and
cachexia (Belter et al., 1968; Johnston et al., 2001a; Morris & Dobson, 2001; Cho et al., 2011).
FEA can metastasize regionally, on the uterine ligament (Papparella & Roperto, 1984), omentum
(Cho et al., 2011), peritoneum (Gelberg & McEntee, 1986) and/or other abdominal organs
(Anderson & Pratschke, 2011; Pires et al., 2012), or at distance, especially to lungs, brain or eyes
(Klein, 2007; Taylor, 2010), causing variable secondary signs, depending on their location.
The clinical history, presenting complaint and physical examination are features used to
diagnose the disease. Vaginal bleeding or hemorrhagic discharge should alert the practitioner to
the possibility of FEA. The differential diagnosis list for hemorrhagic discharge should include
abortion, uterine tumor, cystic endometrial hyperplasia and pyometra. Thereafter, the physical
exam can use abdominal palpation, radiological exploration and/or ultrasound to identify the
tumor in the uterine horns and to search for metastasis. Usually however, FEA is diagnosed after
9
OVH or necropsy, if performed (Belter et al., 1968; Morris & Dobson, 2001; Klein, 2007;
Saraiva et al., 2012). Only histology provides a definitive diagnose of this tumor (Klein, 2007).
1.3.4 Treatment and prognosis
The supportive treatment varies with the stage of the neoplasm when diagnosed and with
additional clinical signs. Supportive care and antibiotics may always be pondered in the presence
of pyometra (Saraiva et al., 2012).
The final treatment should be surgery. OVH is recommended whenever the uterus shows
abnormal macroscopic features, and the abdominal cavity should be explored for any metastatic
foci, that should be removed and sent with the entire surgical specimen of the uterus for
histopathologic analysis(Saraiva et al., 2012).
Despite being largely untested in veterinary medicine, chemotherapy and radiation can be
used with the surgery, when malignancy is detected by histopathology (Morris & Dobson, 2001),
as long the owner is familiar with the unclear benefits of it. This adjunctive treatment may
prevent or delay the development of metastasis. Health of the patient should be monitored in
follow-up appointments every three months to control undesirable effects (Saraiva et al., 2012).
Because of the nonspecific signs and metastatic potential of the FEA, the initial prognosis
should be initially guarded. The presence of metastasis demands a grave prognosis (Saraiva et
al., 2012). Most cases involve a late diagnosis that resulted in a poor prognosis and, in some
cases, euthanasia (Preiser, 1964; Belter et al., 1968; Sapierzynski et al., 2009; Anderson &
Pratschke, 2011). In a study by Miller and collaborators (2003), four of eight diagnosed cases
had metastized and only two survived longer than 5 months. However, a favorable prognosis can
be given when a tumor is detected early, and surgery is performed. Furthermore, non-invasive
tumors usually tend to have a better prognosis, but a long-term evaluation time is warranted
(Saraiva et al., 2012).
1.4 Tumor-suppressor mechanisms
Cancer is a progressive process that arises from a continuum where somatic cells acquire
activating (oncogenes) or deactivating (tumor suppressor genes) mutations. In a series of welldefined steps, the mutations overcome the barriers that normally restrain uncontrolled cellular
growth (Coussens & Werb, 2001; Vesely et al., 2011). Fortunately, numerous intrinsic and
extrinsic tumor-suppressor mechanisms exist to prevent tumor development. Many of these
processes had never been visualized in vivo, but have been inferred by experimental methods.
10
The most important experiments have compared tumor evolution in immune suppressed and
normal mice (Kim et al., 2007; Teng et al., 2008; Hanahan & Weinberg, 2011; Vesely et al.,
2011).
The cancer immunosurveillance hypothesis, first conceived by Paul Ehrlich (at end of
nineteenth century), proposed that the immune system could repress the incidence of neoplasia.
However, it was only in the mid twentieth century, after studies on allograft rejection in which
immune cells had a key role, that the Ehrlich’s idea returned(Dunn et al., 2002). Initially, Ehrlich
proposed that “small accumulations of tumor cells may develop, and because of their possession
of new antigenic potentialities, they provoke an effective immunological reaction with regression
of the tumor and no clinical hint of existence”(Burnet, 1957). However, this idea was then put
aside in the late seventies of last century due to the difficulty in showing its existence in
experimental animals (Thomas, 1982; Dunn et al., 2002). Even Hanahan & Weingerg, who
published an important article in 2000 where they listed six basic alterations essential for
malignant growth, did not include the role of the immune system in the development of cancer
(Hanahan & Weinberg, 2000; Dunn et al., 2002; Cavallo et al., 2011). It was only after the work
of other authors and the discoveries of NK cells, interferon γ and their functions, and also
various studies with inbred mice that two hallmark observations related to the role of the immune
system were added (Smyth et al., 2000; Dunn et al., 2002; Cavallo et al., 2011; Hanahan &
Weinberg, 2011; Vesely et al., 2011), that highlighted the role of the immune system in the
occurrence and evolution of the tumor.
1.4.1 Intrinsic mechanisms
The fundamental mechanisms of cellular division and DNA replication carry the inherit
danger that the replication machinery will inevitably make mistakes. Therefore, intrinsic tumorsuppressor mechanisms, triggering senescence or apoptosis and repairing genetic mutation, will
attempt to repair and prevent the acquired capability of cells to proliferate without environmental
cues acting as a barrier to the further development of any preneoplasic cell (Hanahan &
Weinberg, 2000; Vesely et al., 2011).
Cellular senescence is characterized by permanent quiescence of the cell-cycle with
specific changes in morphology and gene expression that can be triggered by activated
oncogenes or induced by numerous cellular proteins. Escaping oncogene-induced senescence is
now considered a prerequisite for cellular transformation and cell immortality (Serrano et al.,
1997; Vesely et al., 2011).
11
Apoptosis is another intrinsic mechanism for preventing tumors. There are two important
ways to promote apoptosis: the intrinsic and extrinsic pathways. The first mechanism includes
the p53 activation, and, having sensed the activity of oncogenes, initiation of programmed cell
death machinery. Another intrinsic pathway includes alterations such as cellular stress, injury or
lack of survival signals or alterations in mitochondria integrity that cause a release of
proapoptotic effectors that trigger the executioner caspases, resulting in cell death. The extrinsic
death pathways are activated through bonding to cell-surface death receptors such as TNFR,
TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-R2 and Fas-CD95 with their
correspondent ligands, inducing the formation of a signaling complex that activates caspase 8,
and initiates the apoptosis caspase cascade (Peter & Krammer, 2003; Vesely et al., 2011).
Despite the importance of these mechanisms, alternative forms of cell death such as
necrosis, autophagy and mitotic catastrophe can halt the transformation process and recently are
receiving increased attention (Danial & Korsmeyer, 2004; Vesely et al., 2011).
1.4.2 Extrinsic mechanisms
The mechanisms that prevent cancer cells from invading and spreading to other sites are
called extrinsic tumor-suppression mechanisms. These mechanisms involve cells sensing that
adjacent tissues have cancerous cells. Three mechanisms are identified. Cells depend on specific
trophic signals in the microenvironment to check their suicidal tendencies. When there is a
failure in these signals, like the disruption of epithelial cell extracellular matrix, cells activate
their death pathway. A second mechanism appears to involve links between cell polarity genes
that control cell junctions and proliferation. A dysregulation of junctional complexes can also
lead to cell death. The last mechanism, and the one more important to the present study,
involves the limitation of transformation or growth of tumor cells by effector cells of the immune
system (Vesely et al., 2011). However, the immune system has complex functions that cannot be
limited to only destroying tumor cells. Humoral factors and cell interactions form a complex and
active relationship between the immune system and tumors, called immunoediting
1.4.2.1
Immunoediting
Vesely et al. (2011) defined immunoediting as a continuous process during tumor genesis
and progression where the immune system both protects against neoplasia development and
promotes their growth.
12
Characterized as a three-phase process, immunoediting integrates one of the extrinsic
tumor-suppression mechanisms. This concept that the immune system recognizes and destroys
tumor cells was conceived 50 to 100 years ago, and despite all the controversy regarding this
idea had over the years, studies supported concept (Smyth et al., 2000; Dunn et al., 2002; Vesely
et al., 2011). Some authors (Hanahan & Weinberg, 2011), defend the importance of the immune
system on tumor initiation and progress, by establishing the capacity of the tumor to avoid
immune destruction and the importance of the tumor-promoting inflammation as two hallmarks
of cancer biology. Both the innate and adaptive immune system are able to contribute
significantly to immune surveillance and tumor eradication (Kim et al., 2007; Teng et al., 2008;
Hanahan & Weinberg, 2011). Studies using immunodeficient and immunocompetent mice,
researchers have shown the importance of both components of the immune system, and the
important role that both these arms of the IS have in tumor immunogenicity (Hanahan &
Weinberg, 2011; Vesely et al., 2011). Tumors are influenced by the immunological environment
in which they form. Highly immunogenic cancer cells clones are destroyed by the IS leaving the
less immunogenic neoplastic clones that have acquired mechanisms to evade or suppress the IS
as, for example, the secretion of TGF-β and other suppressive factors that may paralyze
infiltrating CTLs and NK cells allowing these clones to grow and form a solid tumor (Dunn et
al., 2002; Hanahan & Weinberg, 2011). Hence, it can be concluded that the immune system
controls both the number of tumor cells and the quality, selecting tumors better suited to survive
in a immunologically intact environment (Vesely et al., 2011).
After multiple iterations of “editing”, a dormant tumor cell population results that can
escape and grow unrestricted by the IS and emerge as a clinically, apparent entity with the IS
acting as a promoter (Hanahan & Weinberg, 2011; Vesely et al., 2011).
For a better understanding of how this concept evolves, an overview of each of these
phases and the mechanisms involved in the operation will follow below (Figure 5).
13
Figure 5. The three phases of cancer immunoediting
Cancer immunoediting is the result of three processes that can function either independently or in sequence to
control and shape cancer. After normal cells are transformed into tumor cells, the immune system can function as
an extrinsic tumor suppressor, eliminating tumor cells or preventing their outgrowth. On elimination phase, also
known as cancer immunosurveillance, innate and adaptive immune cells and molecules recognize and destroy
transformed cells, resulting into a return to normal physiological tissue. Though, if antitumor immunity is unable
to eliminate transformed cells, surviving variants may enter into an equilibrium phase, where cells and molecules
of adaptive immunity prevent tumor outgrowth. These variants may, eventually, acquire further mutations,
resulting into the evasion of tumor cell recognition, killing or control by immune cells, leading them to progress to
clinically detectable malignancies in the escape phase (Vesely et al., 2011).
1.4.2.1.1 Elimination phase
Immunosurveillance is the primary process occurring in the first phase of cancer
immunoediting. The innate and adaptive immune systems locate, recognize and destroy, or at
least, select transformed cells, resulting a phenotypic non-diseased tissue or leading to an
equilibrium phase with better adapted tumor clones (Dunn et al., 2002; Vesely et al., 2011).
14
1.4.2.1.2 Equilibrium phase
This phase is characterized by tumor dormancy, where any tumor cell clone that survived
the elimination process, after acquiring a means of evading immune-mediated recognition and
destruction, remains in patients for decades but may eventually recur as local lesions or as distant
metastases. When antitumor immunity is unable to eliminate completely transformed cells, the
tumor enters an equilibrium phase, where the IS and the tumor find a dynamic balance (Dunn et
al., 2002; Vesely et al., 2011).
1.4.2.1.3 Escape phase
Contrary to the immunosurveillance and equilibrium, that is usually clinically silent the
escape phase represents a dramatic result of cancer immunoediting, where the IS fails either to
eliminate or control transformed cells, and allows surviving and modified tumor cell variants to
grow in an immunologically unrestricted manner, having circumvented both innate and adaptive
immunological defenses. Moreover, the IS contributes to tumor progression by selecting more
aggressive tumor variants through constant immunological pressure and suppressing the
antitumor immune response for modifications effected by the tumor in immune cells or
promoting tumor cell proliferation (Dunn et al., 2002; Dunn et al., 2004; Dunn et al., 2006;
Vesely et al., 2011).
1.4.2.2
Inflammation and cancer
Inflammation is a complex physiological process in that the IS destroys a stressor and
restores the tissue homeostasis (Medzhitov, 2008). Although widely recognized now, one of the
first references to a dense infiltrate of leukocytes in tumors may have been made by Virchow in
1860´s (Sica et al., 2006; Jin et al., 2010; Hanahan & Weinberg, 2011; Vesely et al., 2011). This
response was initially thought to be an attempt of the host to eradicate the tumor (de Visser et al.,
2006; Hanahan & Weinberg, 2011). Population-based studies have shown that individuals with
unresolved, chronic inflammation disease have an increased risk of developing cancer (Mauad et
al., 1994; de Visser et al., 2006) and many tumors are believed to be developed in consequence
of infectious conditions (Pikarsky et al., 2004; de Visser et al., 2006; Rakoff-Nahoum, 2006;
Correa & Houghton, 2007; Gonzalez et al., 2012), indicating an important relationship between
inflammation and tumor genesis. In recent years, there has been increasing evidence for a second
immune system-related hallmark of cancer, tumor-promoting inflammation (Hanahan &
Weinberg, 2011). Although inflammation has a role in tumor elimination, tumors can also recruit
15
immunosuppressive inflammatory cells and create an inflammatory microenvironment
promoting tumors progression (Hanahan & Weinberg, 2011; Vesely et al., 2011). Some studies
performed on mice actually emphasized the importance of the IS in tumor progression and
evolution, showing that the IS can have a role on tumor prevention but at other times is a major
tumor promoter (de Visser et al., 2006; Hanahan & Weinberg, 2011; Vesely et al., 2011).
The microenvironment is important for the tumor. Even without external stimuli, tumor
cells have the ability, through oncogene-driven signals, to activate intrinsic pro-inflammatory
pathways (Cavallo et al., 2011). Necrosis has a key role in inflammation, since necrotic cells
release pro-inflammatory signals to the surrounding microenvironment, recruiting inflammatory
cells (Galluzzi & Kroemer, 2008; Grivennikov et al., 2010; White et al., 2010). All these proinflammatory mechanisms can lead to the presence of pro-tumor chronic inflammatory factors
instead of an antitumor inflammatory response (Mauad et al., 1994; Smyth et al., 2000; de Visser
et al., 2006). In fact, chronic inflammation is considered important in promotion of cellular
proliferation and cancer progression by enhancing angiogenesis and tissue invasion (Vesely et
al., 2011) and release of reactive oxygen species, products promoting carcinogenesis in nearby
cells and accelerating genetic mutations through a state of malignancy (Hanahan & Weinberg,
2011). Finally, through cancer-derived products, immune and regulatory cells are recruited that
weaken tumor antigenicity and subvert immune cells to ultimately promote cancer progression
(Hagemann et al., 2008; Vesely et al., 2011).
In conclusion, one understands the words of Hanahan (2011), “inflammation can be
considered an enabling characteristic for it contributions to the acquisition of core hallmark
capabilities”.
1.4.2.2.1 Macrophages
Macrophages are multifunctional cells. They sense and kill invading microorganisms;
remove dead, dying and damaged cells; promote the bond between innate and acquired immune
responses by releasing cytokines; present antigens to T cells; stimulate B and other cells; and
have a major function in tissue repair (Mantovani et al., 2002; Sica et al., 2008; Tizard, 2012).
Monocytes mature to macrophages when they migrate to the tissues. But, unlike monocytes that
are found only in blood and serous cavities, macrophages are found spread through all body
tissues. Their names differ depending on their location in tissues (Tizard, 2012). However, they
belong to the mononuclear phagocyte system, a plastic and versatile cell lineage recognized as
having their response affected by microenvironmental influences and leading to the expression
of distinct functions (Mantovani et al., 2002; Tizard, 2012).
16
Two subpopulations of macrophages, M1 and M2, are recognized as having a dual
function in their interaction with neoplasic cells, depending whether they are activated
(Mantovani et al., 2002; Tizard, 2012). The macrophages residing in tumor sites are collectively
termed tumor associated macrophages (TAMs), and are considered a major component of
infiltrating leukocytes making a significant component of cancer-associated inflammation
(Mantovani et al., 2002; Jin et al., 2010). These cells originate from blood monocytes recruited
from tumor vasculature and, with a strong relation with B and T lymphocytes, can be activated
as pro-inflammatory or anti-inflammatory cells, depending on the microenvironmental signals
(Sica et al., 2008).
M1 or classically activated macrophages (CAM) are pro-inflammatory cells with
important roles in the defense of the organism. In the presence of a tumor, these cells are the
main responsibles for their elimination (Mantovani et al., 2002; Sica et al., 2006; Karp &
Murray, 2012). Their major aptitude is to provide defense against foreign pathogens and
coordinate local immune cells diapedesis, contributing to a balance between antigen availability
and clearance, through phagocytosis and degradation of neoplasic cells (Sica et al., 2008), acting
thus as anti-tumor cells. Produced early on in the inflammatory process, the differentiation into
these cells depends on the production of gamma interferon (INF-γ) by natural killer cells (NK)
(O'Sullivan et al., 2012; Tizard, 2012), leading them to become NO producers, an important
citotoxic agent, and allowing them to become tumor suppressors (Tizard, 2012).
Eventually, M1 macrophages, in later stages and under the influence of tumor-inhibition
to macrophage activation, acquire the properties of a polarized M2 phagocyte population, also
called alternatively activated macrophages (AAM) (Mantovani et al., 2002; Sica et al., 2006;
Whiteside, 2006; Sica et al., 2008; Jin et al., 2010; Tizard, 2012), who scavenge debris, regulate
wound healing, drive fibrosis and suppress inflammation (Mantovani et al., 2002; Sica et al.,
2008; Karp & Murray, 2012; O'Sullivan et al., 2012; Tizard, 2012). They are considered the
active cells in tumor progression and invasion, due to their action, that leads to important
modifications on the relationship between the immune system and the tumor (Sica et al., 2008;
Tizard, 2012). Through their polarization, M2 macrophages display poor antigen presenting
capacity and induce the presence of T regulation lymphocytes (Treg) that suppress T effectors
cells and monocytes, leading to a restraint of the adaptive and innate immunity. They accumulate
preferentially in poorly vascularized regions of the tumor, with poor oxygenation, inducing the
neo-angiogenesis ability in M2 macrophages even as the synthesizing of chemokines, Matrixmetalloproteinases (MMPs) and TGFβ, lead to a matrix remodeling. Also, the chemokines and
17
MMPs, with tumor necrosis factors (TNF), promote tumor invasion and metastasis. Finally, the
production of these growth factors and inhibition of NO secretion induce the tumor growth and
survival (Mantovani et al., 2002; Sica et al., 2006; Tizard, 2012).
There is a relationship between the improvement of intra-tumor macrophages and the
upgrading of vessel density and tumor progression, showing that macrophages are active players
in the process of tumor survival, progress and invasion (Figure 6) (Sica et al., 2006; Sica et al.,
2008; Vesely et al., 2011), being associated to poor prognosis in several tumors, (Jin et al.,
2010).
Figure 6. TAMs pro-tumoral functions.
Tumor-associated macrophages (TAM) display several pro-tumoral functions. Chemokines have a
prominent role as they induce neo-angiogenesis, activate matrix-metalloproteases (MMPs) and stroma
remodelling, and direct tumor growth. Selected chemokines and immunosuppressive cytokines inhibit
the anti-tumor immune response (Sica et al., 2006).
1.4.2.2.2. Lymphocytes
In the primary response, the acquired immune system usually takes several days to
become effective against a specific antigen. However, the specific immune system have the
capacity of memory, and in subsequent contact with the same agent (on the secondary response),
the antibody and cell answer quickly recognize the antigen and make a faster and more effective
response. Each of these different responses is the responsibility of two different groups of cells:
T and B lymphocytes. Lymphocytes are small, round cells, with 7-15 µm with a large round
nucleus that stains intensively with hematoxylin. Usually localized in lymphoid organs, blood
and related to mucosal surface, these uniform appearance cells are a mix of subpopulations that
can only be properly identified and distinguished through their behavior, cell surface proteins
18
and products (Goldsby et al., 2000; Tizard, 2012). Despite these two major types of lymphocytic
population, the NK cells could be considered as a granular lymphocyte that belongs to the innate
immune system and are considered to have a key role in the relationship between immune
system and tumors (Tizard, 2012).
Tumor-infiltrating lymphocytes (TILs) are the cells inside the group of lymphocytes that
are commonly correlated with tumor disease and prognosis, varying its importance to the group
taken into consideration (Vanherberghen et al., 2009; Carvalho et al., 2011). Also, is of general
knowledge that different lymphocytes have different functions and that, through their
permanence on tumor microenvironment they can be functionally compromised (Whiteside,
2004; Whiteside, 2006). An example of this loss of functionality is the failure of the T-cell
receptor-associated signaling pathway, making them unable to successfully exercise the
fundamental molecular pathway that leads to their activation against a cognate antigen
(Whiteside, 2004).
1.4.2.2.2.1.
T lymphocytes
T lymphocytes have recently been a main target of interest in tumor inflammation, being
referred to in association with different types of tumors, with the aim of becoming a new
therapeutic weapon (Zhang et al., 2003; Macchetti et al., 2006; Sheu et al., 2008; Tomsova et
al., 2008; Leffers et al., 2009; Carvalho et al., 2011).
T cells are the main mediated immunity effectors and are matured on the thymus from
bone marrow precursors. Each T cell is programmed to recognize a specific antigen through their
T cell receptor (TCR). However, a T cell only recognizes antigens when they are presented by an
antigen presenting cell (APC), in the context of their major histocompatibility complex (MHC)
proteins, meaning that it cannot be activated through soluble antigens. This receptor is intimately
related to a group of molecules only expressed by T lymphocytes, the CD3. For that reason,
these molecules became a useful marker for these types of cells (Ferrer et al., 1993; Goldsby et
al., 2000; Tizard, 2012). The major APC are the dendritic cells that could activate the naïve T
cells, and the macrophages that can only activate the previously triggered T lymphocytes
(Tizard, 2012).
Besides the MHC, and CD3 proteins, T cells also have other molecules on their surface
that act as co-receptors and that allow them to be divided into two major different
subpopulations: the CD4 and CD8 molecules. The CD4 is present in T helper lymphocytes,
which only recognize antigens linked to class II MHC molecules and that also have regulatory
functions, while CD8 is on the surface of T lymphocytes which have cytotoxic and suppressor
19
functions and only recognize antigens linked to class I MHC molecules (Goldsby et al., 2000;
Tizard, 2012). The ratio CD4/CD8 can vary with the type of tumor, and some studies correlate a
high tumor content of CD8 with a better prognosis, as T lymphocytes are able to destroy tumor
cells (Carvalho et al., 2011). However, in some cases, the recruitment of Tregs by the tumor can
cause a functional paralysis of cytotoxic T cells, allowing the tumor to progress (Whiteside,
2006; Vesely et al., 2011).
1.4.2.2.2.2.
B lymphocytes
Few B cells circulate in the blood. They are mainly found in the cortex of lymph nodes,
in marginal zones in the spleen, in the bone marrow, throughout the intestine and Peyer´s
patches, and in mucosal associated lymphoid tissues (MALT). This subtype of lymphocytes
produces antibodies that bind and destroy exogenous antigens. Tumor cells are antigenic and
stimulate the cell-mediated immune response. Antibodies to tumor cells can be found in many
tumor-bearing animals. Along with complement, these antibodies can lyse free tumor cells in the
bloodstream, but are not effective in destroying the cells in solid cancers. However, antibodies
can have an opposite effect, as blocking antibodies may be produced. As non-complementactivating, antitumor antibodies can bind and mask tumor antigens on the cell surfaces,
protecting them from the attack of cytotoxic T cells (Goldsby et al., 2000; Tizard, 2012).
Furthermore, although not well studied and being considered uncommon, plasma cells
can be found in tumors (Kornstein et al., 1983). Although the antibodies are usually synthesized
by B cells in other organs, like lymph nodes, spleen and liver, the presence of these cells on the
tumor is being explained as a humoral response to tumor neo-antigens (Hansen et al., 2001;
Coronella et al., 2002; Nzula et al., 2003) and may have an important biological action, as these
antibodies may be tumor-specific and bind a intracellular protein translocated and presented to
the cell surface upon tumor cell apoptosis (Hansen et al., 2001). However, the biological
significance and prognostic values of B-TILs are still unknown, despite the ability to make
antibodies in situ which may be an important aspect of host defense (Whiteside, 2006). These
cells have been well studied in virus induced tumors like mouse mammary tumor virus, where B
cells’ stimulation would assure the maintenance and possibly the amplification of the virus and
increase the probability of infecting the mammary gland (Held et al., 1993).
20
Chapter 2 - Objectives
With this project we intend to:

Optimize immunohistochemistry technique for the identification of macrophages
and lymphocytes (B and T-cells) in the feline tissues;

Quantify feline endometrial macrophage, T-lymphocytes and B-lymphocytes
populations on control tissues (healthy uterus in follicular and luteal phases of the
reproductive cycle);

Quantify macrophages, T-lymphocyte and B-lymphocyte populations in feline
endometrial adenocarcinomas and on the tissue neighboring the tumor;

Statistically compare this population of cells on the non-diseased (control) feline
endometrium and on feline endometrial adenocarcinomas.
21
Chapter 3 – Material and methods
3.1 Biological material
Material for study was from the archive of the Anatomical Pathology and Histology
Laboratory of Universidade de Trás-os-Montes e Alto Douro (LHAP-UTAD). These samples
came from several veterinary practices. The anonymity of the sample sources was respected.
Ten samples of feline papillary serous adenocarcinomas were studied (Table 1). Five of these
also had pyometra. Another twenty uteri (ten in follicular and ten in luteal phase) were studied
as controls.
Table 1. Description of the used tumor cases
Assembling of the available clinical data of the studied animals. (y = years, m = months, U = unknown).
Cases
1
2
3
Breed
European
Shorthair
European
Shorthair
European
Shorthair
Age
12 y
U
U
Morphology Pyometra
Papillary
serous
Papillary
serous
Papillary
serous
Myometrium
Contraception
Invasion
Clinical
signs
Yes
Yes
U
U
Yes
Yes
U
U
Yes
No
U
Vulvar
discharge
Bloody
vulvar
discharge
Bloody
vulvar
discharge
4
European
Shorthair
2y
Papillary
serous
Yes
No
No
5
European
Shorthair
8m
Papillary
serous
Yes
No
No
No
Yes
No
U
No
No
U
U
No
Yes
U
U
No
Yes
U
U
No
No
U
U
6
7
8
9
10
European
Shorthair
European
Shorthair
European
Shorthair
European
Shorthair
European
Shorthair
9y
5y
8y
10 y
7y
Papillary
serous
Papillary
serous
Papillary
serous
Papillary
serous
Papillary
serous
The uteri had been fixed in 4% buffered formaldehyde immediately after surgery and sent
to LHAP. The non-diseased uterus samples were collected cranially at 1cm from the ovary and
caudally at 1cm from the uterine body in both uterine horns. Ovaries collected with the uterus
were used to estimate the phase of estrus. In samples from diseased uterus, the organ was cut
sagittally to better perceive the extent of the tumor. After identifying areas of endometrial
22
hyperplasia and areas of tumor development, samples were harvest from these last areas. All
samples were then routinely paraffin-embedded and cut in 3 µm sections.
3.2 Sample selection
For sample selection, 3 µm sections stained in haematoxylin-eosin were used. The nondiseased cyclic samples were staged as follicular or luteal phase upon the endometrial
morphology (number and coiling of the endometrial glands) and the type and dimensions of
ovarian structures (follicles vs. corpora lutea).
Furthermore, tumor samples were also routinely stained in haematoxylin-eosin and
analysis being performed by two different pathologists. They were classified according to the
type of feline endometrial adenocarcinoma proposed by Saraiva et al. (2012).
3.3 Immunohistochemistry analysis
The selected samples were then submitted to indirect immunohistochemistry technique to
identify the different types of immune cells. Briefly, the 3 µm tissue sections were mounted on
silane-coated slides (3-aminopropyltriethoxysilane, Sigma®), deparaffinized in xylene and
rehydrated in a graded alcohol series, ending with tap water. They were then subjected to thermal
treatment (three cycles of five minutes in the microwave oven at 750W) in citrate buffer. After
cooling (30 minutes) at room temperature, samples were dipped in hydrogen peroxide at 3% for
30 minutes. After incubation in the normal serum, the slides were then incubated with the
primary antibody that was diluted in a solution of PBS and BSA (Sigma®), ranging the dilution
expressed in Table 2. They were then subjected to the biotinilated serum, to the enzymatic
complex and to DAB for revelation. Gill´s hematoxylin was used as counterstain, and after
dehydrated, the slides were mounted with a lamella, using Entelan® (Merck) to get a definitive
preparation.
All immunohistochemistry analyze were performed along with a positive and negative
control. For positive control a feline lymph node was used. Negative controls were performed
replacing the primary antibody for PBS and BSA on a positive lymph node.
For macrophages the chosen molecule was the MAC387 antibody, that binds to the LI
protein, which is also present in the cytoplasm of resting peripheral neutrophils and monocytes
(Brandtzaeg et al., 1988). This antibody was showed to have cross reactivity with the cat (Obert
& Hoover, 2002).
23
CD3 is one chain of the T-cell receptor, and was shown to be a very reliable antibody in
the identification of these cells (Alibaud et al., 2000).
The CD79a is one of the two polypeptide chains of the CD79 molecule, physically
associated with the membrane with immunoglobulin, present in a wide range of mature B-cells
(Mason et al., 1995).
All used clones of antibodies showed crossed reactivity with cat tissue.
Table 2. Used immunohistochemestry technic’s specifications
Resume of the used antibodies as well as the differences in the technic used for each one.
Cells type
Antibody/clone
Macrophages
T Lymphocytes
Mac 387 (Serotec®)
CD 3 (Dako®)
B Lymphocytes
CD 79 (Cell
Marque®)
UltraVision TM Detection System (Thermo
Incubation method
NovoLink® Max
Fisher Scientific, LabVision Corporation,
Polymer Detection
Fremont, CA, USA)
System (Leica)
Primary antibody
incubation
Dilution
Over-night
2 hour
1:100
1:50
1:100
Bovine serum albumin
(Sigma®)
50%
100%
DAB (NovoLink®
Revelation
Max Polymer
DAB (Sigma®) for 10 minutes
Detection System,
Leica) for 30 minutes
3.4 Quantification
The positive cells presented a distinct brownish to gold labeling in the membrane or
cytoplasm and a corresponding morphological aspect to the cells in study.
24
In non-diseased (control) tissue samples the positive cells were counted in the 10 fields
on superficial layer and in 10 fields on the deep layer of the endometrium (Figure 7). The
counted area was chosen randomly and without overlap.
Figure 7. Definition of layers on non-diseased uterus
Definition of the two considered layers of the endometrium (in purple the surface layer and
in orange the deep layer) in both follicular (A) and luteal (B) phases.
Bar=300micra
In tumor samples the hot spots areas were privileged on the different layers and also in
the surrounding tissue (Figure 8), 10 fields each. Similarly to the non-diseased uterus samples,
not only was a superficial area chosen near the lumen, but also a deep layer of the tumor near the
myometrium.
Figure 8. Definition of layers on tumor
Definition of the considered layers on the tumor: A – Surface layer;
B – Deep Layer; C – Surrounding tissue
Bar=300micra
25
The analysis of the surrounding tissue compromised 10 images, against 20 of the tumor.
For that, the values obtained from cells counting on this layer we compared to the tumor by
evaluating their means.
The fields were photographed using a microscope, NIKON Eclipse E600® (Nikon
Instruments Europe BV, Kingston, Surrey, UK), that capture digital images using a NIKON
Digital Camera DXM200® and NIKON ACT-1® (version2.12) imaging software with the
objective of 40X.
Using Adobe Photoshop® (version CS5) a patronized grid with 0, 04 mm2 was adapted to
each captured image and every cell present with positive staining was counted.
The results were then assembled in a Excel®, Office 2007 table and statistically treated.
3.5 Statistical Analysis
Statistical comparisons were performed using the IBM SPSS Statistics Base 19.0
statistical software for Windows, using descriptive statistics, analysis of variance (ANOVA) and
means compared by Tukey Post Hoc tests to identify the differences between layers and cycle
phases and tumors. We took into account the variations that could be promoted by the presence
of pyometra or myometrium invasion. A p value ≤0.05 was regarded as statistically significant.
26
Chapter 4 – Results
4.1. Macrophages
The results obtained for macrophages showed some differences between layers in each
phase, between oestrous cycle phases and between non-diseased tissues and the tumor.
Figure 9. Macrophages’ immunohistochemistry results
Results of positive staining for Mac 387 on surface follicular endometrium (A), deep luteal endometrium
(B) and tumor surface (C) and deep layer (D).
Bar = 30micra
Macrophages in non-diseased endometrium - despite no significant differences were
observed between layers in each cycle phase, in the luteal period the difference is close to the
statistical significant level (p=0,064) (Attachment 1). Also, we can observe a decrease in cell
counting mean value on surface layer between follicular and luteal phase and an increase in their
mean values on the deep layer. Lastly, it can be observed that the deep luteal layer shows a larger
27
standard error than on other layers, showing more variable values of macrophages (Table 3 and
Graphic 1).
Macrophages on tumors – the number of macrophages is higher on tumors than on the
non-diseased uterus (whatever phase or layer in comparison), and also have higher standard
errors than the non-diseased uterus (Table 3 and Graphic 1). Tumors with pyometra showed
significant differences (p<0,05) both to non-diseased uterus as to tumor without pyometra
(Attachment 2). In case of myometrium invasion no significant changes were observed on the
number of cells studied (Graphic 1 and Attachment 9).
Macrophages on peripheral tissue – comparing the adjacent tissues infiltrate with the
macrophages inside the tumor, the ratio of cells is maintained and no significant differences were
observed (Attachment 8 and Graphic 2). The presence of pyometra significantly increases
(p=0,008) the numbers of infiltrating macrophages in peripheral tissue (Attachment 7 and
Graphic 1). Despite no significant differences were observed (Attachments 9 and 10), in absolute
terms, we can see higher numbers of macrophages on peripheral tissue when the tumor didn’t
invade the myometrium (Graphic 1).
Table 3. Descriptive analysis of macrophages results
Results for the descriptive statistics for macrophages infiltration in the feline
endometrial stroma in cyclic, non-diseased and in endometrial adenocarcinoma samples.
Samples
Follicular
Cyclic
Luteal
Without pyometra
Tumors
With pyometra
Layer
X
SD
Min
Max
Median
Supf
9,6
4,01
3
16
9
Deep
7,2
4,29
3
17
6,5
Supf
4,2
3,36
0
9
4
Deep
16,8
19,9
1
57
6
Supf
46,2
14,48
31
63
52
Deep
44,6
24,3
12
75
43
Periph
23
17,8
1
44
22
Supf
129,2
101,5
40
259
73
Deep
124
103,3
27
293
83
Periph
129
64,5
36
196
140
X - Mean; SD – Standard deviation; Min – minimum; Max – Maximum; CV – Coefficient of variation; Supf – Superficial;
Periph – Peripheral
28
Graphic 1. Macrophages results
Comparison of macrophages counting values between layers. A – Follicular phase (n=10); B – Luteal
phase (n=10); C – Tumor without pyometra (n=5); D – Tumor with pyometra (n=5); E – Comparison
between tumor mean and peripheral layer without pyometra (n=5); F – Comparison between tumor
mean and peripheral layer with pyometra (n=5); G – Comparison between tumor mean and
peripheral layer without myometrium invasion (n=5); H – Comparison between tumor mean and
peripheral layer with myometrium invasion (n=5).
The values of tumor mean and peripheral tissue, without great differences between them, increase
greatly when pyometra is present (note the scale diference between A and B and C and D).
29
4.2. B lymphocytes
The results obtained for B lymphocytes also showed differences between layers, among
the different phase of oestrous cycle and comparing non-diseased uterus to tumors.
Figure 10. B lymphocytes’ immunohistochemistry results
Results of positive staining for CD79a on surface follicular endometrium (A, circle), deep luteal
endometrium (B) and tumor surface (C) and deep layer (D).
Bar = 30micra
B cells in non-diseased endometrium - the results presented significant differences
between both layers on luteal uterus (p=0,004) (Attachment 3). A reduce occurrence of B cells in
both surface and deep layer can be observed together in follicular and luteal phase, with a small
reduction of standard error on deep layer (Table 4 and Graphic 3).
B cells on tumors – On tumors studied, the frequency of B cells were differently marked
when pyometra was present or absent (Table 4 and Graphic 3). The tumor without pyometra
showed no significant differences on B cell infiltrate when compared to non-diseased uterus, but
when in the presence of this inflammation, the occurrence of these cells increases greatly
(Attachment 4). There are no differences when comparing the surface and deep layer on tumor
30
with or without pyometra, and both values have higher standard errors than the non-diseased
uterus (Attachment 3). No significant differences were observed whether there is or not
myometrium invasion (Graphic 2 and Attachment 9).
B cells on peripheral tissue – Comparing the adjacent tissues infiltrate with the B
lymphocytes inside the tumor, the ratio of cells is maintained and no significant differences were
observed (Attachment 8 and Graphic 4). The presence of pyometra significantly increases
(p=0,046) the numbers of infiltrating B lymphocytes in the peripheral tissue (Attachment 7 and
Graphic 4). Despite no significant differences were observed (Attachments 9 and 10), in absolute
terms we can see a reduction of B lymphocytes both on the tumor and peripheral tissue when
invasion is present (Graphic 2).
Tabel 4. Descriptive analysis of B lymphocytes results
Results for the descriptive statistics for B lymphocytes infiltration in the feline endometrial stroma in
cyclic, non-diseased and in endometrial adenocarcinoma samples.
Samples
Follicular
Cyclic
Luteal
Without pyometra
Tumors
With pyometra
Layer
X
SD
Min
Max
Median
Supf
6,1
3,1
2
11
6,5
Deep
8,7
6,83
2
20
6,5
Supf
3
2,62
1
7
1,5
Deep
7,6
3,57
1
12
8,5
Supf
19
8,5
10
32
19
Deep
11,2
3,19
7
15
12
Periph
17,6
9,37
7
29
20
Supf
109,4
83,39
37
239
83
Deep
233,6
264
81
700
107
Periph
230,2
201,3
60
576
186
X - Mean; SD – Standard deviation; Min – minimum; Max – Maximum; CV – Coefficient of variation; Supf – Superficial;
Periph – Peripheral.
31
Graphic 2. B lymphocytes results
Comparison of B lymphocytes counting values between layers. A – Follicular phase (n=10); B –
Luteal phase (n=10); C – Tumor without pyometra (n=5); D – Tumor with pyometra (n=5); E –
Comparison between tumor mean and peripheral layer without pyometra (n=5); F – Comparison
between tumor mean and peripheral layer with pyometra (n=5); G – Comparison between tumor
mean and peripheral layer without myometrium invasion (n=5); H – Comparison between tumor
mean and peripheral layer with myometrium invasion (n=5). (note the scale difference between
different graphics).
32
4.3. T lymphocytes
The count of T lymphocytes infiltrate showed differences between layers, concerning the
oestrous cycle and between the control uterus and the tumors studied.
Figure 11. T lymphocytes’ immunohistochemistry results
Results of positive staining for CD3 on surface follicular endometrium (A), deep luteal endometrium (B)
and tumor surface (C) and deep layer (D).
Bar = 30micra
T cells on non-diseased endometrium - The results of T cell counting present significant
differences between both layers in the follicular phase (p=0,001), being more numerous on the
surface layer (Table 5). Also, a lower number of T cells were observed on the surface layer in
luteal uterus relating to follicular phase, and the inverse when comparing the deep layer (Table 5
and Graphic 5). In luteal uterus, the T cells evaluations had a higher standard error than the
follicular phase (Table 5).
33
T cells on tumors – The mean value of T cell infiltration on endometrial tumors
significantly increased (p<0,05) when compared to the non-diseased uterus, being very similar
despite the presence or absence of pyometra (Attachment 6 and Graphic 5). The values show no
significant differences between layers in both tumor situations, and both have higher standard
errors than the non-diseased uterus (Table 5 and Attachment 5). No significant differences were
observed whether there is or not myometrium invasion (Graphic 3 and Attachment 9).
T cells on peripheral tissue – The adjacent tissues shows higher T cell infiltration
whether pyometra is present or absent, but no significant differences between them were
observed (Attachment 7 and 8 and Graphic 3). Despite no significant differences were observed
(Attachments 9 and 10), in absolute terms we can see a reduction of T lymphocytes both on the
tumor and peripheral tissue when invasion of myometrium is present. Also, cells counting was
higher on the peripheral tissue than in the tumor (Graphic 3).
Table 5. Descriptive analysis of T lymphocytes results
Results for the descriptive statistics for macrophages infiltration in the feline endometrial stroma in
cyclic, non-diseased and in endometrial adenocarcinoma samples.
Samples
Follicular
Cyclic
Luteal
Without pyometra
Tumors
With pyometra
Layer
X
SD
Min
Max
Median
Supf
44,9
11,1
33
68
41,5
Deep
25
9,96
9
43
24,5
Supf
26,3
30,32
3
99
11
Deep
38,7
34,56
10
122
22,5
Supf
159,8
128,9
48
332
101
Deep
74,8
46,6
33
147
58
Periph
151,2
128,2
36
342
115
Supf
157,4
59,79
75
214
182
Deep
93
70
37
210
67
Periph
173,6
94,4
82
308
180
X - Mean; SD – Standard deviation; Min – minimum; Max – Maximum; CV – Coefficient of variation; Supf – Superficial;
Periph – Peripheral
34
Graphic 3. T lymphocytes results
Comparison of B lymphocytes counting values between layers. A – Follicular phase (n=10); B –
Luteal phase (n=10); C – Tumor without pyometra (n=5); D – Tumor with pyometra (n=5); E –
Comparison between tumor mean and peripheral layer without pyometra (n=5); F – Comparison
between tumor mean and peripheral layer with pyometra (n=5); G – Comparison between tumor
mean and peripheral layer without myometrium invasion (n=5); H – Comparison between tumor
mean and peripheral layer with myometrium invasion (n=5). (Note the scale difference between
different graphics).
35
Chapter 5 – Discussion
Most authors consulted consider FEA as an uncommon lesion (Preiser, 1964; Kenedy et
al., 1998; Miller et al., 2003; Saraiva et al., 2012). Indeed, most published studies include few
cases (Preiser, 1964; Miller et al., 2003; Gil da Costa et al., 2009). In cattle and rabbits, uterine
adenocarcinoma is considered more common (Preiser, 1964; Kenedy et al., 1998). Rabbits does
have a 79% of incidence of uterine adenocarcinoma after 5 years of age. The fact that they are
induced ovulators and, many times, housed individually, can presumably explain this high
incidence (Elsinghorst et al., 1984). Similarly, virgin Han:Winstar rats, have a 39% incidence of
uterine adenocarcinoma, and these rats also are subjected to long periods of estrogenic stimuli
(Deerberg et al., 1981). With a seasonal cycle and induced ovulation, the cat also is also
subjected to long periods of estrogenic influence. Thus, early ovariohysterectomy, is considered
protective against FEA, and the practice of breeding or inducing artificial ovulation (that reduce
the exposure to estrogen) in these animals could explain the low incidence reported (Stabenfeldt
& Pedersen, 1991; Saraiva et al., 2012).
Recently, FEA was diagnosed more frequently in Portugal and was described to be 20%
of incidence in all cases of uterine lesions observed by Saraiva et al. (2012). These differences
between countries may be explained by husbandry differences. In Portugal, queens are spayed
often after the first heat or only when there is genital disease while in other countries, OVH is
done before the first heat (Saraiva et al., 2012). This practice could explain differences in FEA
development. In addition, clinical signs of FEA are non-specific, increasing the chance of
confusing FEA with other´s uterine pathologies, especially pyometra (Saraiva et al., 2012). In
published case reports, few detail the clinical aspects of the FEA (Preiser, 1964; Belter et al.,
1968; Anderson & Pratschke, 2011; Cho et al., 2011). The infrequency on uterus examination
(Gelberg & McEntee, 1986), the lack of interest on the anatomopathological evaluation of
ovariohysterectomy surgical specimens, most of them related with pyometra, an inadequate post
mortem evaluation or even the fact that some small masses in the uterus can be overlooked on a
standard analysis can explain this low number of cases (Saraiva et al., 2012). Miller and
collaborators (2003) considered the uterus the site where genital tumors occur most commonly
in cats. Recently, the LHAP, where this work was done, systematically harvested specimens
from spayed queens and performed histopathological analysis, and around a 20% incidence of
FEA was described (personal communication from Maria dos Anjos Pires - LHAP).
Other feline genital tumors are more common and leiomyoma, is considered the most
common mesenchymal tumor on the feline uterus (McEntee & Nielsen, 1976; Papparella &
36
Roperto, 1984; McEntee, 1990). Leiomyomas are firm, opalescent to tan nodules in the
myometrium. They have interwoven bundles of leiomyocytes and fibroblasts with uncommon
mitotic figures (McEntee, 1990; Kenedy et al., 1998). Despite not being considered a neoplasic
lesion, adenomyosis is common in the bitch, cow and queen must be considered a differential
diagnosis for FEA invasion. This frequent canine and feline lesion (Gelberg & McEntee, 1986;
Bernardo, 2012) could be concurrent with FEA. This lesion, often a nodular growth of
endometrial glands forming cysts filled with mucus or neutrophils may pre-exist in pyometras
and commonly extends to the serosa (McEntee & Nielsen, 1976; Kenedy et al., 1998). Also, the
cystic endometrial hyperplasia, a frequent lesion in a dogs (Payan-Carreira & Pires, 2005), is
referred in a cat (Bernardo, 2012) and could be concurrent with FEA. Other lesion present on the
uterus is the endometrial polyps. These pedunculated proliferative growths protruding on uterus
lumen were considered non-preneoplastic (Gelberg & McEntee, 1984). Despite this, Saraiva et
al. judged that the luminal papillary growths supported by a fibrovascular stroma and having
anisokaryosis, anisocytosis, bizarre mitotic figures and numerous multinucleated cells, in some
cases invading the myometrium, were FEA.
The mammalian endometrium is a dynamic and complex tissue that functions to
guarantee embryo implantation, survival and maintenance of pregnancy. Cyclic remodeling of
the uterus is a response to sex steroids and controlled by several factors including cytokines,
interleukins and growth factors. Between functional and structural cells, immune cells are found
in the endometrium. Their recruitment has been proved to be cycle dependent, under steroid
influence, participating with stromal and epithelial cells in the regulation of the cyclic
remodeling and embryo implantation (Payan-Carreira et al., 2011). Some regulatory mechanisms
of immune cells in the uterus have already been studied in different species, and the macrophage
is one of the best studied immune cells (De & Wood, 1990; Butterworth et al., 2001; Kaeoket et
al., 2001; Gu et al., 2005). Butterworth et. al.
(2001) studied inflammatory cells in the
endometrium on four non-diseased queens without specification of the estrous cycle. Also,
unlike this thesis, the entire wall of the reproductive tract was studied without the diferentiating
structures and layers in the uterus and the cell markers used were different from the ones used in
this study. This thesis, to our knowledge, is the first study of immune cells along the estrous
cycle on the endometrium and in FEA. These reasons turn this into a groundbreaking work.
As the number and frequency of immune infiltrate changes between species (Pires et al.,
2006), the comparison of our results with other species is not the most suitable. However, taking
into account the physiological aspects of each species and knowing the IS and the cells, one
37
might infer and compare the results with others species like the rat, sow and the woman (De &
Wood, 1990; Kaeoket et al., 2001; Jones et al., 2004; Gu et al., 2005). This work presents
differences on immune cell endometrial distribution during estrous phases. The differences were
not statistically significant, but this may be due to a low sample number or, in some cases, due to
individual physiologic variation.
T lymphocytes were more common than other cell types, consistent with the findings of
Butterworth et al. (2001) that showed the CD8+ T lymphocytes were the most common type of
immune cell in the feline reproductive tract. The reduction of macrophages and T cells in the
surface layer on the luteal phase could be explained by uterine preparation for embryo
implantation that takes place on this phase. Localized immunodepression may be necessary to
allow acceptance of the fetal “allograft.” Macrophages and T cells increased in the deep layer of
the luteal phase. This could indicate a migration of these cells to allow the implantation of the
embryo (Kaeoket et al., 2001; Kayisli et al., 2004; Gu et al., 2005; Payan-Carreira et al., 2011).
The variation between macrophages and T cells was to some degree predictable physiologically.
Both estrogen and progesterone have an important, and as yet unexplained, role on macrophage
distribution (De & Wood, 1990). Conversely, B cells decreased in both layers as queens changed
from the follicular to the luteal phase, perhaps in response to the same mechanism that reduced
macrophages and T lymphocytes. However, this doesn´t explain the reduction of B cells in the
deep layer where macrophages and T cells increased. Because the luteal phase is longer in the
feline reproductive cycle and sperm could be found or pseudopregnancy can develop, the luteal
samples could be less homogeneous than the follicular. Therefore, more cell number variation
could be expected in the luteal phase. However, macrophages of the luteal surface layer and B
cells in both luteal layers decreased when compared with the follicular phase. The low sample
number could explain these results.
The IS is important in the tumor biology. In early tumor development, the IS acts to
eliminate the tumor, but in the following phase, an equilibrium is established with immune cells
(especially macrophages and T cells) facilitating tumor growth metastasis (Vesely et al., 2011).
Knowing the kinetics of tumor development, early increase of immune cells in a tumor would be
expected when compared with the non-diseased (control) uterus.
This study showed macrophages increased in the tumors, suggesting their importance on
the tumor biology. However, macrophages subsets were not evaluated. Subset identification is
important since the M1 macrophages are related with tumor elimination, and M2 subpopulation
promote tumor progression (Mantovani et al., 2002; Sica et al., 2008). Thus, it would be
38
interesting to compare population numbers or their ratio and distribution between FEA and nondiseased endometrial tissue. Likewise only total number of T lymphocytes were evaluated, but
how T-cell subsets changed in tumors and non-diseased uteri and how they reflect survival and
tumor progression or elimination (Whiteside, 2006; Tizard, 2012) would be informative.
Regardless, T cells are an important cell in FEA suggesting that T-cells were stimulated by
tumor antigens. Unlike T-cells and macrophages, B-cells may not have so great importance on
FEA biology. Although a small increase of B-cells were seen in FEA when compared with nondiseased uterus, the increase was not statistically significant. Actually, there is little knowledge
and understanding of the relation between B-cells and tumors (Nzula et al., 2003). Some studies
of breast cancer suggest the possibility that most of the antibodies produced to breast tumors and
regional ganglions are likely to be against auto-antigens (Coronella et al., 2002). Never the less,
it remains hard to explain the differences of infiltrating B-cells between FEA and other
neoplasms studied, such as breast adenocarcinoma, where up to 20% have a large infiltration of
B-cells (Balch et al., 1990).
Finally, differences observed between layers on the tumor might be related to a loss of
stratification noticed in these cases. The lost of organized endometrial strata may induced local
increase of immune cells related with the deregulation of concentrations or stimulating factors of
tumor regulation and inflammation.
The pyometra and FEA are commonly seen together. However it is not known which
appears first, the pyometra or the FEA. Chronic inflammation is often seen with tumors. Indeed,
the action of immune cells, through reactive oxygen and nitrogen species, induce DNA damage
on proliferating cells, and repeated tissue damage and regeneration could result in genomic
mutations and promote the development of tumor cells (Coussens & Werb, 2002). This causal
relationship of inflammation and pathology has been shown to take part in colon carcinogenesis
and in synovial arthritis (Yamanishi et al., 2002). Also, Th lymphocytes, recruited by tumor cells
could reduce the presence of effector cells on tumor (Vesely et al., 2011).
Interpretation of the relationship of immune cells present on FEA with or without
pyometra was not one of the objectives of this work. Pyometra was associated with increased of
macrophages and B-cell. Only after the study of the subsets of macrophages populations, could it
be discussed if the increase in macrophages is related with the tumor, the infection or both
processes. It would be interesting to compare the changes in the pattern of distribution of
macrophage subtypes to understand how neoplastic responses change in infection. B-cells
increased in FEA with pyometra, seen mainly as plasma cells. The presence of these plasma cells
39
on tumors that present pyometra could reflect a variation of local antigen stimulation and
inflammatory regulators. On other hand, T-cells in FEA with or without pyometra did not change
significantly, suggesting that T-cell chemotactic stimuli were the same in both circumstances.
However, T-cell subsets changes that might have occurred would not be noticed because of the
limitations of the study. Different subtypes of T-cells, also would have shown the types related to
pyometra versus neoplasia (Tizard, 2012). The inflammatory/infection stimulus may have
decreased the regulatory cells stimulated by the tumor or changed the overall organization of
these cells subtypes in the tumor.
The study of the immune cells infiltrating tissue outside of the tumor (in this specific
case, on the myometrium surrounding the endometrial lesion) attempted to understand how much
the infiltrate was due to tumor versus infection as we had no pyometra available as control.
The three cell types increased in the presence of the tumor, when compared with nondiseased tissue. However, it was found that numbers of macrophages and B-cells when FEA isn’t
associated with pyometra were low when compared with the presence of inflammation
(pyometra). T cells increases at the periphery of the tumor and in both cases were not
significantly different.
In the end, only by evaluating subsets of inflammatory cells and immunomodulating
proteins can one really understand these cells variations among non-diseased, tumor and
pyometra.
The lack of clinical history and follow-up of the studied animals limited our evaluation of
the cyclic evaluation to the ovarian and uterine microscopic morphology. Likewise it limited
conclusions on the tumors. It would be important to correlate the presence of immune cells with
the surveillance of the FEA, and the follow-up to correlate to our results, given a prognostic
value to the immune cells ratios. Several studies have been presented correlating the presence
and numbers of a certain type of immune cell with the prognosis of the tumor. Jochems &
Schlom (2011) resume several studies made in humans, where immune system may or may not
be an important and independent prognostic factor for several types of tumors. Ino and
collaborators (2008) showed that a reduction of TILs and NK cells in endometrial endometrioid
adenocarcinoma can contribute to the disease progression. Also, de Jong and collaborators
(2009) showed that the presence of high numbers of CD8+ T-lymphocytes was an independent
prognostic factor in endometrial cancer. However, most of these studies focus in only one type of
cell, and some of the comparisons didn´t have non-diseased tissues as a control.
40
The relation between the IS and the tumor is complex and involves different cells,
extracellular matrix, biological and molecular compounds that change with the tumor. Hence, the
evaluation of only one type of immune cell (or even three types) may be incomplete. By
comparing the cells inside tumors with apparently healthy tissues in the same animal should not
be considered as representative because all tissues may suffer the influence of the stimulus that
the tumor generates, thus skewing values. Perhaps, studying cell ratios and comparing them with
the values in similar tissues of non-diseased animals might be a more accurate approach to this
research. Additionally, our results were influenced by the low number of cases. Although the
twenty non-diseased samples used for evaluation of the endometrium and ten FEA (the highest
number of FEA cases thus far studied), the differentiation between the cases that presented with
and without pyometra reduced our statistical conclusions. In future, we would like to
complement the non-diseased samples with a group of animals in anestrus, a complex stage that
in Portugal is limited to a short period of December.
As we referred before, the lack of clinical data and follow up of the studied cases, don’t
allow us to have predictive studies as the prognostic value of our data. The myometrium invasion
is a severe feature, morphologically related with a poor prognostic, instead the lack of
information that we have on the disease evolution. In the work presented to this thesis, some
cases (5/10) had myometrium invasion. Along the cell infiltrate evaluation, and despite the lack
of significance of our results, some variances between the numbers of cells counting could be
observed. In fact, when myometrium invasion is noted, we can observe a reduction of the values
on peripheral tissue for all cells types and a reduced number of B and T lymphocytes inside the
tumor. A future study, with more complete clinical information and higher number of cases
could allow a better understanding of the relation between the myometrium invasion and the
immune system, and if the variations of the numbers of these type of cells could be proposed as a
prognostic factor.
The choice to study these three immune cells was attempted to get a broad feeling for
their importance in the non-diseased uterus and is this unstudied tumor. However, future
identification of subtypes and other cells important on tumor development and elimination will
provide information on the true importance and function of immune cells on the non-diseased
uterus and its tumors. While the present study did not give an explanation of B-cell functions
there is a dearth of work on B-cell roles in the reproductive tract or in FEA.
41
Chapter 6 - Final considerations
Reviewing this thesis, one feature that limited our discussion and possible conclusions
about the importance of the immune cells on these tumors was the limited number of samples
and lack of clinical history, aspects out of our control.
However, the objectives were accomplished. An unquestionable identification of the
three studied immune cells was achieved with the chosen antibodies, although validation for two
antibodies in the cat was not available (for B and T-cells).
Also, infiltrating macrophages, B-cells and T-cells were quantified in both non-diseased
and tumor tissue. This allowed us to confirm and understand their distribution on non-diseased
tissue during follicular and luteal stages of the reproductive cycle and compare them with that of
the FEA.
In non-diseased uterus, it was possible to observe an interesting change of distribution of
the studied cells, with the tendency to decrease their numbers on the surface of the endometrium
and increase them on deep layer of luteal stage, when compared with the follicular stage.
Indeed, macrophages and T-cells may be important in the development of FEA. Further
studies should be done to better understand their function and importance. The presence of
pyometra was associated with an increase of B-cells and, to a lesser extent, of macrophages in
the neoplasm. There does not seem to be an increase of immune cells in peripheral tissue unless
pyometra is present.
The presence of myometrium invasion is related with all cell types reduced numbers in
the peripheral tissues of the tumor and with only maintenance of the amount of macrophages
within the tumor.
For these reasons, we conclude that this innovator study provided information to drive
future studies on this aspect of tumor immunobiology, the relation between the IS, the feline
reproductive cycle and FEA biology.
More complete series could allow us to see if the evaluation of immune cells could be
related and proposed as a prognostic value.
42
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49
Chapter 8 – Attachments
Attachment 1. Analysis of variance of macrophages results
Comparison between variance values of both layers on reproductive cycle and tumor with and without pyometra.
No significant differences observed between layers. p value ≤0.05
ANOVA
Surface X Deep layer
Sum of
Cycle Vs Tumor
Squares
Follicular
Between Groups
Luteinic
Tumor without pyometra
Tumor with pyometra
df
Mean Square
28,800
1
28,800
Within Groups
310,000
18
17,222
Total
338,800
19
Between Groups
793,800
1
793,800
Within Groups
3669,200
18
203,844
Total
4463,000
19
6,400
1
6,400
Within Groups
3200,000
8
400,000
Total
3206,400
9
67,600
1
67,600
Within Groups
83858,800
8
10482,350
Total
83926,400
9
Between Groups
Between Groups
Attachment 2. Post Hoc test to macrophages results
p value ≤0.05 and means for groups in homogeneous subsets are
displayed. Significant differences observed between tumor with
pyometra and other groups.
Contagem
Tukey HSD
a,b
Subset for alpha = 0.05
Cat_Ciclo
N
1
2
Follicular
20
8,4000
Luteinic
20
10,5000
Tumor without pyometra
10
45,4000
Tumor with pyometra
10
Sig.
126,6000
,097
50
1,000
F
Sig.
1,672
,212
3,894
,064
,016
,902
,006
,938
Attachment 3. Analysis of variance of B lymphocytes results
Comparison between variance values of both layers on reproductive cycle and tumor with and without pyometra.
Significant values between layers in luteal phase. p value ≤0.05
ANOVA
Contagem
Sum of
Fasextumor
Folicullar
Squares
Mean Square
33,800
1
33,800
Within Groups
507,000
18
28,167
Total
540,800
19
Between Groups
105,800
1
105,800
Within Groups
176,400
18
9,800
Total
282,200
19
Tumor without
Between Groups
152,100
1
152,100
pyometra
Within Groups
330,800
8
41,350
Total
482,900
9
38564,100
1
38564,100
Within Groups
306600,400
8
38325,050
Total
345164,500
9
Luteinic
Tumor with pyometra
Between Groups
df
Between Groups
Attachment 4. Post Hoc test to B lymphocytes results
p value ≤0.05 and means for groups in homogeneous subsets
are displayed. Significant differences observed between tumor
with pyometra and other groups.
Contagem
Tukey HSD
a,b
Subset for alpha = 0.05
Fasextumor
N
1
2
Luteal
20
5,3000
Follicular
20
7,4000
Tumor without pyometra
10
15,1000
Tumor with pyometra
10
Sig.
171,5000
,988
51
1,000
F
Sig.
1,200
,288
10,796
,004
3,678
,091
1,006
,345
Attachment 5. Analysis of variance of T lymphocytes results
Comparison between variance values of both layers on reproductive cycle and tumor with and without pyometra.
Significant values between layers in follicular phase. p value ≤0.05
ANOVA
Contagem
Cicloxtumor
Sum of Squares
Folicullar
Luteinic
Tumor With Pyometra
Mean Square
Between Groups
1980,050
1
1980,050
Within Groups
2008,900
18
111,606
Total
3988,950
19
768,800
1
768,800
Within Groups
19020,200
18
1056,678
Total
19789,000
19
Between Groups
18062,500
1
18062,500
Within Groups
75105,600
8
9388,200
Total
93168,100
9
Between Groups
10368,400
1
10368,400
Within Groups
33923,200
8
4240,400
Total
44291,600
9
Between Groups
Tumor Without Pyometra
df
Attachment 6. Post Hoc test to T lymphocytes results
p value ≤0.05 and means for groups in homogeneous subsets are
displayed. Significant differences observed between control uterus
and tumors.
Contagem
Tukey HSD
a,b
Subset for alpha = 0.05
Cicloxtumor
N
1
2
Luteinic
20
32,5000
Folicullar
20
34,9500
Tumor Without Pyometra
10
117,3000
Tumor With Pyometra
10
125,2000
Sig.
,999
52
,981
F
Sig.
17,742
,001
,728
,405
1,924
,203
2,445
,157
Attachment 7. Analysis of variance of immune cells counting on peripheral tissue whether
pyometra is or not present
Comparison between values of peripheral layer’s immune cells counting with and without the
presence of pyometra. Significant differences observed on peripheral layer of B lymphocytes
values between tumors with or without pyometra present. p ≤0.05.
ANOVA
Cell
Layer
LinfB
peripheral
LinfT
Mac
peripheral
peripheral
Sum of Squares
df
Mean Square
Between Groups
112996,900
1
112996,900
Within Groups
162448,000
8
20306,000
Total
275444,900
9
1254,400
1
1254,400
Within Groups
101350,000
8
12668,750
Total
102604,400
9
Between Groups
28090,000
1
28090,000
Within Groups
17918,000
8
2239,750
Total
46008,000
9
Between Groups
F
Sig.
5,565
,046
,099
,761
12,542
,008
Attachment 8. Analysis of variance of immune cells counting between tumor and peripheral
tissue considering pyometra
Comparison between values of tumor and peripheral layer’s immune cells counting with and
without the presence of pyometra. No significant differences observed. p ≤0.05.
ANOVA
Cell
Pyometra
LinfB
with
without
LinfT
with
without
Mac
with
without
Sum of Squares
Between Groups
df
Mean Square
8614,225
1
8614,225
Within Groups
236186,300
8
29523,288
Total
244800,525
9
15,625
1
15,625
Within Groups
467,900
8
58,488
Total
483,525
9
5856,400
1
5856,400
Within Groups
48674,000
8
6084,250
Total
54530,400
9
2873,025
1
2873,025
Within Groups
93267,100
8
11658,388
Total
96140,125
9
14,400
1
14,400
Within Groups
56284,700
8
7035,588
Total
56299,100
9
Between Groups
1254,400
1
1254,400
Within Groups
1920,700
8
240,088
Total
3175,100
9
Between Groups
Between Groups
Between Groups
Between Groups
53
F
Sig.
,292
,604
,267
,619
,963
,355
,246
,633
,002
,965
5,225
,052
Attachment 9. Analysis of variance of immune cells counting on tumor mean and peripheral tissue
whether there is or not myometrium invasion
Comparison of tumor mean and peripheral layer’s immune cells counting with and without the presence
myometrium invasion. No significant differences observed. p ≤0.05.
ANOVA
Cell
Layer
LinfB
mean
peripheral
LinfT
mean
peripheral
Sum of Squares
Between Groups
mean
peripheral
Mean Square
20250,000
1
20250,000
Within Groups
115108,600
8
14388,575
Total
135358,600
9
27562,500
1
27562,500
Within Groups
247882,400
8
30985,300
Total
275444,900
9
9090,225
1
9090,225
Within Groups
31656,900
8
3957,113
Total
40747,125
9
Between Groups
19536,400
1
19536,400
Within Groups
83068,000
8
10383,500
102604,400
9
324,900
1
324,900
Within Groups
56446,100
8
7055,763
Total
56771,000
9
Between Groups
12673,600
1
12673,600
Within Groups
33334,400
8
4166,800
Total
46008,000
9
Between Groups
Between Groups
Total
Mac
df
Between Groups
54
F
Sig.
1,407
,270
,890
,373
2,297
,168
1,881
,207
,046
,835
3,042
,119
Attachment 10. Analysis of variance of immune cells counting between tumor and peripheral tissue
tissue considering invasion
Comparison between values of tumor mean and peripheral layer’s immune cells counting with and without the
presence myometrium invasion. No significant differences observed. p ≤0.05.
ANOVA
Sum of
Cell
Invasion
LinfB
Not present
Present
LinfT
Not present
Present
Mac
Not present
Present
Squares
Between Groups
df
Mean Square
3629,025
1
3629,025
Within Groups
329407,500
8
41175,938
Total
333036,525
9
1334,025
1
1334,025
Within Groups
33583,500
8
4197,938
Total
34917,525
9
7617,600
1
7617,600
Within Groups
81239,400
8
10154,925
Total
88857,000
9
1836,025
1
1836,025
Within Groups
33485,500
8
4185,688
Total
35321,525
9
2449,225
1
2449,225
Within Groups
42539,000
8
5317,375
Total
44988,225
9
6579,225
1
6579,225
Within Groups
47241,500
8
5905,188
Total
53820,725
9
Between Groups
Between Groups
Between Groups
Between Groups
Between Groups
55
F
Sig.
,088
,774
,318
,588
,750
,412
,439
,526
,461
,516
1,114
,322