| Abstract | Istraživani su tumori cirkumanalnih žlijezda s ciljem pronalaženja imunohistokemijskih
markera za bolje razlučivanje i karakteriziranje histoloških tipova ovih tumora.
S istim ciljem se primjenjivala i metoda izračuna fraktalne dimenzije. Odabrano je ukupno
54 tumora cirkumanalnih žlijezdi (15 adenoma, 11 epitelioma, 21 dobro diferencirani
karcinom te 7 slabo diferenciranih karcinoma), kao negativna kontrola korišteno je
10 normalnih cirkumanalnih žlijezdi izuzetih od pasa bez patoloških promjena na
cirkumanalnim žlijezdama. Za provjeru koreliranosti sa kliničkim ponašanjem tumora
izvršena je anketa vlasnika pasa sa kojih su uklonjeni navedeni tumori. Nakon histološke
klasifikacije po smjernicama WHO, učinjena je imunohistokemijska pretraga sljedećim
biljezima: vimentin, E-kadherin, N-kadherin, matriksalna metaloproteinaza-9, Ki-67, estrogen
receptor α i androgen receptor. Iste su i procijenjene („skorirane“) uobičajenim metodama.
Reakcije vimentinskim biljegom su fotografirane te su iste nakon pretvorbe u crno-bijele slike
upotrijebljene za izračunavanje fraktalne dimenzije primjenjujući „box counting“ metodu
izračuna i računalni program „Fractalyse“. Rezultati imunohistokemijske analize ukazuju na
signifikantne razlike između tipova tumora ovih žlijezdi kada se radi o vimentinu te Ki-67.
Konkretno, u slučaju vimentina najsignifikantnije se razlikuju skupine benignih tumora
i normalnih žlijezdi u odnosu na maligne tumore (tu su ubrojeni i epiteliomi). Vrijednost
ekspresije Ki-67 rasla je sa rastom histološke malignosti tumora, te je ona iznosila 5,67% u
normalnih žlijezda, Istraživani su tumori cirkumanalnih žlijezda s ciljem pronalaženja imunohistokemijskih
markera za bolje razlučivanje i karakteriziranje histoloških tipova ovih tumora.
S istim ciljem se primjenjivala i metoda izračuna fraktalne dimenzije. Odabrano je ukupno
54 tumora cirkumanalnih žlijezdi (15 adenoma, 11 epitelioma, 21 dobro diferencirani
karcinom te 7 slabo diferenciranih karcinoma), kao negativna kontrola korišteno je
10 normalnih cirkumanalnih žlijezdi izuzetih od pasa bez patoloških promjena na
cirkumanalnim žlijezdama. Za provjeru koreliranosti sa kliničkim ponašanjem tumora
izvršena je anketa vlasnika pasa sa kojih su uklonjeni navedeni tumori. Nakon histološke
klasifikacije po smjernicama WHO, učinjena je imunohistokemijska pretraga sljedećim
biljezima: vimentin, E-kadherin, N-kadherin, matriksalna metaloproteinaza-9, Ki-67, estrogen
receptor α i androgen receptor. Iste su i procijenjene („skorirane“) uobičajenim metodama.
Reakcije vimentinskim biljegom su fotografirane te su iste nakon pretvorbe u crno-bijele slike
upotrijebljene za izračunavanje fraktalne dimenzije primjenjujući „box counting“ metodu
izračuna i računalni program „Fractalyse“. Rezultati imunohistokemijske analize ukazuju na
signifikantne razlike između tipova tumora ovih žlijezdi kada se radi o vimentinu te Ki-67.
Konkretno, u slučaju vimentina najsignifikantnije se razlikuju skupine benignih tumora
i normalnih žlijezdi u odnosu na maligne tumore (tu su ubrojeni i epiteliomi). Vrijednost
ekspresije Ki-67 proliferacijskog indeksa potvrđuju nedavne spoznaje o ovom protutijelu kao dobrom biljegu
razlika u proliferativnoj sposobnosti ovih tumora i žlijezdi općenito. |
| Abstract (english) | Introduction
Tumors of circumanal glands are among most common in dogs. Mostly, these are
benign tumors – adenomas. However there is also a considerable number of carcinomas
and epitheliomas. Differences in biologic behaviour of epitheliomas and carcinomas, just as
histopathologic caracterization, profiles of several interesting immunohistochemical stainings
are not so far precisely defined or known. This investigation aims at revealing differences
in features of benign and malignant types of circumanal gland tumors using several
immunohistochemical stainings and determination of fractal dimension (FD).
Materials and methods
A total of 54 circumanal gland tumors were chosen from the archives of the
Department of Veterinary Pathology, University of Zagreb. Precise number of samples
was as follows: 15 adenomas, 11 epitheliomas, 21 well differentiated carcinomas, 7 poorly
defferentiated carcinomas. A control of 10 samples of normal circumanal gland obtained from
necropsies were also included. After precise histologic classification according to WHO,
an immunohistochemical staining with seven different markers was performed. Used IHC
markers were: vimentin, E-cadherin, N-cadherin, matrix metalloproteinase 9, Ki-67, estrogen
receptor α and androgen receptor. The IHC reactions of mentioned markers were scored
using conventional methods. Additionaly, vimentin IHC reactions were photographed at
two different magnifications -100X and 400X and converted in black and white images thus
enhancing the positive vimentin reactions. These images were used for determination of FD
applying „box counting method“ and computer software „Fractalyse“. Independently, in order
to gain clinical information regarding individual tumor behaviour (reoccurrence of tumors
or metastasis, survival after initial diagnosis etc.), a telephone survey of dog owners that
were included in the study was conducted. To determine significance of results, conventional
statistics were used using Microsoft Excel and Statistica software.
Results
IHC staining showed differences considering the tumor type. Above all this was
pronounced in Introduction
Tumors of circumanal glands are among most common in dogs. Mostly, these are
benign tumors – adenomas. However there is also a considerable number of carcinomas
and epitheliomas. Differences in biologic behaviour of epitheliomas and carcinomas, just as
histopathologic caracterization, profiles of several interesting immunohistochemical stainings
are not so far precisely defined or known. This investigation aims at revealing differences
in features of benign and malignant types of circumanal gland tumors using several
immunohistochemical stainings and determination of fractal dimension (FD).
Materials and methods
A total of 54 circumanal gland tumors were chosen from the archives of the
Department of Veterinary Pathology, University of Zagreb. Precise number of samples
was as follows: 15 adenomas, 11 epitheliomas, 21 well differentiated carcinomas, 7 poorly
defferentiated carcinomas. A control of 10 samples of normal circumanal gland obtained from
necropsies were also included. After precise histologic classification according to WHO,
an immunohistochemical staining with seven different markers was performed. Used IHC
markers were: vimentin, E-cadherin, N-cadherin, matrix metalloproteinase 9, Ki-67, estrogen
receptor α and androgen receptor. The IHC reactions of mentioned markers were scored
using conventional methods. Additionaly, vimentin IHC reactions were photographed at
two different magnifications -100X and 400X and converted in black and white images thus
enhancing the positive vimentin reactions. These images were used for determination of FD
applying „box counting method“ and computer software „Fractalyse“. Independently, in order
to gain clinical information regarding individual tumor behaviour (reoccurrence of tumors
or metastasis, survival after initial diagnosis etc.), a telephone survey of dog owners that
were included in the study was conducted. To determine significance of results, conventional
statistics were used using Microsoft Excel and Statistica software.
Results
IHC staining showed differences considering the tumor type. Above all this was
pronounced in vimentin stain, which was significantly more expressed in malignant types
of circumanal gland tumors. Ki-67 proliferative index showed also significant differences between benign and malignant types of circumanal gland tumors, namely the values for
indivudual groups (diagnoses) were as follows: normal gland – 5,67% , adenoma – 4,89%,
epithelioma – 14,44%, well differentiated carcinoma – 16,44%, poorly differentiated
carcinoma – 20,76%. Staining of matrix metalloproteinase 9 and estogen receptor α were
showed significant differences only when comparing group of normal circumanal glands
and adenomas versus group consisted of epitheliomas, well and poorly differentiated
carcinomas. E-cadherin showed very high staining level disregarding the tumor type (or
normal circumanal gland). The same is true for N-cadherin, with important difference that this
expression was at all time very low. Androgen receptor showed great variety of expression
within each of investigated group, but failed to show any significant differences between any
tumour types or normal glands.
Values of determined FD varied between different circumanal tumors and normal
glands. They were as follows: normal circumanal glands - 1,318 (1,372), adenomas - 1,384
(1,408), epitheliomas - 1,547 (1,597), well differentiated carcinomas - 1,569 (1,607),
poorly differentiated carcinomas - 1,679 (1,723); [first value is FD determined from 100X
magnification micrographs, second (in brackets) is one determined at 400X magnification
micrographs]. After application od HSD Tukey test, significant differences (p< 0,05)
were determined individually between normal glands or adenomas and epitheliomas, well
differentiated carcinomas and poorly differentiated carcinomas. Simuntaneously, Pearson
correlation between fractal dimensions and type of circumanal tumor (or normal gland) were
between 0,65 and 0,70 for both dimensions (100X and 400X).
Results of telephone survey failed to show any significant difference or corellation
between tumor behaviour and tumor type, value of FD or staining of applied IHC markers.
Discussion and conclusions
Above results indicate vimentin IHC staining and determination of FD as a ancillary
diagnostic methods of choice when discerning between benign and malignant tumors of
circumanal glands. Additional development of method of FD determination may yield a
possibility for this tool to discern within benign and malignant circumanal tumors. Ki-67
staining confirms recent reports which show similar results, and indicates significantly higher
Ki-67 proliferation index in malignant circumanal tumors.
We suggest for future studies involving FD determination in circumanal tumors a
more prospective clinical monitoring of dogs with these tumors to avoid problems regarding
retrospective surveys of the owners. |
