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Effects of electrochemotherapy with cisplatin and peritumoral IL-12 gene electrotransfer on canine mast cell tumors: a histopathologic and immunohistochemical study

Claudia Salvadori

Claudia Salvadori

Department of Veterinary Sciences, University of PisaTuscany, Italy
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Salvadori, Claudia
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Tanja Svara

Tanja Svara

Institute of Pathology, Forensic and Administrative Veterinary Medicine, Veterinary Faculty, University of LjubljanaLjubljana, Slovenia
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Svara, Tanja
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Guido Rocchigiani

Guido Rocchigiani

Department of Veterinary Sciences, University of PisaTuscany, Italy
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Rocchigiani, Guido
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Francesca Millanta

Francesca Millanta

Department of Veterinary Sciences, University of PisaTuscany, Italy
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Millanta, Francesca
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Darja Pavlin

Darja Pavlin

Veterinary Faculty, Clinic for Companion Animals, University of LjubljanaLjubljana, Slovenia
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Pavlin, Darja
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Maja Cemazar

Maja Cemazar

Institute of Oncology LjubljanaLjubljana, Slovenia
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Cemazar, Maja
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Ursa Lampreht Tratar

Ursa Lampreht Tratar

Institute of Oncology LjubljanaLjubljana, Slovenia
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Lampreht Tratar, Ursa
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Gregor Sersa

Gregor Sersa

Institute of Oncology LjubljanaLjubljana, Slovenia
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Sersa, Gregor
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Natasa Tozon

Natasa Tozon

Veterinary Faculty, Clinic for Companion Animals, University of LjubljanaLjubljana, Slovenia
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Tozon, Natasa
 und 
Alessandro Poli

Alessandro Poli

Department of Veterinary Sciences, University of PisaTuscany, Italy
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Poli, Alessandro
  
14. Sept. 2017
Effects of electrochemotherapy with cisplatin and peritumoral IL-12 gene electrotransfer on canine mast cell tumors: a histopathologic and immunohistochemical study's Cover Image
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Artikel-Kategorie: Research Article
Online veröffentlicht: 14. Sept. 2017
Seitenbereich: 286 - 294
Eingereicht: 12. Juli 2017
Akzeptiert: 10. Aug. 2017
DOI: https://doi.org/10.1515/raon-2017-0035
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© 2017 Claudia Salvadori, Tanja Svara, Guido Rocchigiani, Francesca Millanta, Darja Pavlin, Maja Cemazar, Ursa Lampreht Tratar, Gregor Sersa, Natasa Tozon, Alessandro Poli
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Histology of tissue samples collected before the combined therapy (T0) and at 4 (T1) and 8 weeks (T2) post-treatment. (A) At T0 sheets of neoplastic cells with abundant cytoplasm containing variable number of metachromatic granules were present in the superficial and deep dermis; (B) At T1 The neoplastic tissue was substituted by a fibrotic tissue associated with scanty inflammatory infiltrates mainly constituted by mononuclear cells; (C) At T2 in dogs with partial response between connective tissue bundles were evident thin aggregates of neoplastic mast cells. Haematoxylin Eosin; bar = 100 mm.
Histology of tissue samples collected before the combined therapy (T0) and at 4 (T1) and 8 weeks (T2) post-treatment. (A) At T0 sheets of neoplastic cells with abundant cytoplasm containing variable number of metachromatic granules were present in the superficial and deep dermis; (B) At T1 The neoplastic tissue was substituted by a fibrotic tissue associated with scanty inflammatory infiltrates mainly constituted by mononuclear cells; (C) At T2 in dogs with partial response between connective tissue bundles were evident thin aggregates of neoplastic mast cells. Haematoxylin Eosin; bar = 100 mm.

Figure 2

Immunoistochemical staining of tissue samples collected at T0 (A, C and E) and, T1 (B, D and E). CD3+ lymphocytes infiltrating the neoplastic tissue at T0 (A) and the fibrotic tissue at T1 (B). Scanty Foxp3+ Treg lymphocytes at the periphery of neoplastic tissue at T0 (C) and in a tissue sample collected at T1 (D). CD68+ macrophages in the neoplastic tissue at T0 (E) and in the fibrotic tissue at T1 (F). Immunohistochemical staining using DAB chromogen and haematoxylin counterstain. Bar = 100 mm.
Immunoistochemical staining of tissue samples collected at T0 (A, C and E) and, T1 (B, D and E). CD3+ lymphocytes infiltrating the neoplastic tissue at T0 (A) and the fibrotic tissue at T1 (B). Scanty Foxp3+ Treg lymphocytes at the periphery of neoplastic tissue at T0 (C) and in a tissue sample collected at T1 (D). CD68+ macrophages in the neoplastic tissue at T0 (E) and in the fibrotic tissue at T1 (F). Immunohistochemical staining using DAB chromogen and haematoxylin counterstain. Bar = 100 mm.

Figure 3

Histogram of number of immune cells in 10,000 µm2 of tissue samples collected al T0, T1 and T2. Slight increase of CD3+ lymphocytes at T1, while macrophages significantly increased at T2 and Treg lymphocytes at T1.
Histogram of number of immune cells in 10,000 µm2 of tissue samples collected al T0, T1 and T2. Slight increase of CD3+ lymphocytes at T1, while macrophages significantly increased at T2 and Treg lymphocytes at T1.

Figure 4

Immunoistochemical staining of tissue samples collected at T0(A, C and E) and , T1(B, D and E). Ki-67+ neoplastic cells at T0(A) and at T1 in a dog with partial response (B). Bcl-2+ neoplastic cells at T0(C) and at T1 in a dog with partial response (D). Microvessels stained using an anti-CD31 primary antibody at T0(E) and in the fibrotic tissue at T1 in a dog with a complete response (F). Immunohistochemical staining using DAB chromogen and haematoxylin counterstain. Bar = 100 mm.
Immunoistochemical staining of tissue samples collected at T0(A, C and E) and , T1(B, D and E). Ki-67+ neoplastic cells at T0(A) and at T1 in a dog with partial response (B). Bcl-2+ neoplastic cells at T0(C) and at T1 in a dog with partial response (D). Microvessels stained using an anti-CD31 primary antibody at T0(E) and in the fibrotic tissue at T1 in a dog with a complete response (F). Immunohistochemical staining using DAB chromogen and haematoxylin counterstain. Bar = 100 mm.

Figure 5

Histogram of microvessel density determineg using both primary antibodies against Factor VIII and CD31positive-cells in 50,000 µm2 of tissue samples and number of Ki-67+ and Bcl-2+ neoplastic cells in 10,000 µm2 of tissue samples collected al T0, T1 and T2. Microvessel density was drastically reduced at T1 and T2. as well as the proliferation activity of neoplastic cells, while Bcl-2 expression was increased at T1.
Histogram of microvessel density determineg using both primary antibodies against Factor VIII and CD31positive-cells in 50,000 µm2 of tissue samples and number of Ki-67+ and Bcl-2+ neoplastic cells in 10,000 µm2 of tissue samples collected al T0, T1 and T2. Microvessel density was drastically reduced at T1 and T2. as well as the proliferation activity of neoplastic cells, while Bcl-2 expression was increased at T1.

Antibodies used in the study

Antibody Specificity Type Species Source Dilution Pretreatment
Anli-humun CD3 Pan-T lymphocytes Policonal Rabbit (A0552) Dako UK Ltd. Ely UK 1:50 Citrate buffer pH6
Anti-human CD20 Pan-B lymphocytes Policonal Rabbit (RB-9013-PO) Thermo Scientific, Chesire, UK 1:400 None
Anti-human Foxp3 T-reg lymphocytes Monoclonal Mouse (7979) Affymetrix eBioscience, san Diego, CA USA 1:100 Triss-EDTA pH9
Anti-human CD68 Macrophages Monoclonal Mouse (PG-M1) Thermo Scientific, Chesire, UK 1:100 Proteinase K
Anti-human Ki-67 Proliferating cells Monoclonal Mouse (610538) BD Biosciences, Wyckoff, NJ, USA5 1:100 Citrate buffer pH6
Anti-human Bcl-2 Antiapoptotic protein Monoclonal Mouse (610538) BD Biosciences, Wyckoff, NJ, USA5 1:100 Citrate buffer pH6
Anti-human Von Willebrand Factor - Endothelial cells Policlonal Rabbit (A0082) Dako UK Ltd. Ely UK 1:300 Citrate buffer pH6
Anti-human CD31 Endothelial cells Monoclonal Mouse (JC70A) Dako UK Ltd. Ely UK 1:100 Citrate buffer pH6
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