1. Spearman’s rank-correlation coefficient (ρ)
AN IMMUNOHISTOCHEMICAL STUDY OF EPITHELIAL-MESENCHYMAL TRANSITION IN FELINE MAMMARY TUMORS
N. Karabolovski*, A. Gudan Kurilj † K. Severin‡, M. Hohšteter†, I.-C. Šoštarić-Zuckermann†, L. Medven†, B. Artuković† and Ž. Grabarević†
*Department of Veterinary Pathology, University St. Climent Ohridski Bitola, Macedonia, †Department of Veterinary Pathology, University of Zagreb, Croatia and ‡ Department of Forensic and State Veterinary Medicine, University of Zagreb, Croatia
Introduction: Epithelial-mesenchymal transition (EMT), as defined by loss of epithelial characteristics and gain of a mesenchymal phenotype, has been reported in human and canine mammary tumors, but similar study in feline mammary tumors has not been performed. EMT can be induced or regulated by various growth and differentiation factors among which TGFβ has received much attention. The aim of our study was to evaluate loss of epithelial and gain of mesenchymal markers in epithelial neoplastic cells of feline mammary tumors and to assess possible correlation with expression of TGFβ.
Table 1: Antibodies and protocols applied for IHC.
Materials and Methods: A total of 27 feline mammary gland hyperplastic and neoplastic lesions were selected from archive of Department of veterinary pathology Zagreb (4 benign lesions, 3 grade I carcinomas, 8 grade II and 12 grade III carcinomas) to evaluate immunohistochemical (IHC) expression of cytokeratin (CK) AE1/AE3, E-cadherin (E-cadh), vimentin (Vim) and TGFβ (Table 1). One-way ANOVA with post hoc Tukey's test and Spearman's rank correlation coefficient were used for statistical analysis.
MARKERS
PRIMARY ANTIBODY
DILUTION
UNMASKING
INCUBATION
EVALUATION
5 fields x 200
Cytokeratin
Clone AE1/AE3
Dako
1:100
Heat induced/ TRS pH6
30 min
% of weak and negative cells + intensity = score
E-cadherin
Clone NCH-38
1:50
Vimentin
Clone V9
% of positive cells + intensity = score
TGF β
TGF β 17
Novocastra
1:20
Heat induced/ TRS pH9
60 min
Table 2: Expression of markers in neoplastic epithelial cells.
Results: Generally, loss of cytokeratin (Fig. 1) and E-cadherin (Fig. 2), and gain of vimentin (Fig. 3) were more frequently observed in carcinomas of higher malignancy grade compared to benign lesions (Table 2). However, the correlation between each marker showed only positive correlation between loss of E-cadherin and gain of vimentin (r=0.69, p<0.05) in grade II carcinomas (Table 3). The expression of TGFβ (Fig. 4) was significantly higher in carcinomas grade II and III compared to grade I (p<0.05) (Fig. 5).
SCORE
Tumor type/ grade
Loss of
Gain of CK
E-cadh
Vim
TGF β
Lobular hyperplasia 1 2
Fibroadenomatous change 3
Carcinoma tubulopapillary/gr. I 4
Intraductal papillary carcinoma/ gr. I
Carcinoma tubular/ gr. II
Carcinoma tubulopapillary/ gr. II 6
Carcinoma tubulopapillary/gr. II 5
Intraductal papillary carcinoma/ gr. II
Carcinoma solid/ gr. II
Carcinoma tubular/ gr. III
Carcinoma tubulopapillary/ gr. III
Carcinoma solid/ gr. III A B C
Fig. 1. Representative findings of IHC with antibody against cytokeratin. A) strong diffuse expression in carcinoma grade I. Loss of CK expression in (B) carcinoma grade II and (C) carcinoma grade III. A B C
Figure 2. Representative findings of IHC with antibody against E-cadherin. A) strong membranous expression in carcinoma grade I. Loss of E-cadherin expression in (B) carcinoma grade II and (C) carcinoma grade III. A B D C
Fig. 3. Representative findings of IHC with antibody against vimentin. A) Positive ductal epithelial cells in fibroadenomatous change.
Gain of vimentin expression in B) carcinoma grade I, C) carcinoma grade II and D) carcinoma grade III.
Figure 5. Expression of TGFβ in mammary tumors.
Table 3. Correlations between epithelial and mesenchymal markers - carcinomas grade II.
Spearman’s rank-correlation coefficient (ρ)
Loss of
Gain of
Cytokeratin
E-cadherin
Vimentin
TGF β
Loss of Cytokeratin -
-0.33
0.07
-0.54
Loss of E-cadherin
0.79a)
-0.26
Gain of Vimentin
-0.23
Gain of TGF β A C B
Fig. 4. Representative findings of IHC with antibody against TGF β in A) normal gland, B) fibroadenomatous change, C) carcinoma grade I, D) carcinoma grade, II E) carcinoma grade III. D E
a) P<0.05
Conclusions: These findings indicate occurrence of characteristic changes suggestive of EMT in feline mammary carcinomas and a possible role of TGFβ in this process.