Vrednotenje postopkov elektrokemoterapije in genskega elektroprenosa plazmidne DNA z zapisom za pasji interlevkin-12 pri zdravljenju spontanih kožnih in oralnih tumorjev pri psih

Milevoj, Nina (author), Tozon, Nataša (mentor)

Namen doktorske naloge je bil vrednotenje postopkov elektrokemoterapije (EKT) in genskega elektroprenosa (GEP) plazmidne DNA z zapisom za pasji interlevkin-12 (IL-12) pri zdravljenju spontanih kožnih in oralnih tumorjev pri psih. S tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo (LC-MS/MS), smo določili koncentracijo bleomicina (BLM) v krvi zdravljenih psov in izračunali farmakokinetične parametre. V krvi zdravljenih psov smo določali subpopulacije imunskih celic (CD4+ in CD8+ limfociti, regulatorni limfociti T – Treg) s pretočno citometrijo. Z imunohistokemičnim barvanjem smo določali izražanje receptorjev in ligandov programirane celične smrti 1 (PD-1 in PD-L1) v tumorjih zdravljenih psov. Da bi ocenili kakovost življenja psov, zdravljenih z EKT in/ali GEP IL-12, smo njihove lastnike povabili k izpolnjevanju Vprašalnika o kakovosti življenja psov in Kratkega testa za ocenjevanje vprašalnika. BLM pri psih je imel monofazno eliminacijsko krivuljo. Povprečni porazdelitveni volumen (Vd) je bil 224,5 ± 75,02 ml/kg, očistek (CL) 7,04 ± 2,05 ml/min/kg in razpolovni čas (t1/2) 22,03 ± 0,88 min. Telesna masa psov ni vplivala na farmakokinetiko BLM. Starejši psi so imeli nižji Vd, na druge farmakokinetične parametre pa starost ni vplivala. Tekom zdravljenja nismo ugotovili značilnih razlik v odstotku CD4+ in CD8+ limfocitov v krvi zdravljenih psov. Ob koncu opazovalnega obdobja je upadel odstotek Treg v krvi zdravljenih psov; spremembe niso sovpadale z odgovorom na zdravljenje. PD-1 je izražalo 95,2 % mastocitomov in 100 % oralnih melanomov, PD-L1 pa so izražali vsi tumorji. Psi s tumorji z višjim izražanjem PD-1 in PD-L1 so imeli značilno krajši povprečni čas preživetja, slabši odgovor na zdravljenje in krajše preživetje brez napredovanja bolezni kot tisti s tumorji z nizkim izražanjem PD-1 in PD-L1. Lastniki so ocenili, da se je kakovost življenja psov po zdravljenju izboljšala. Bolje so ocenili kakovost življenja pri psih, zdravljenih z neinvazivno vrsto zdravljenja, pri tistih, ki so se na zdravljenje odzvali, in pri tistih z manjšimi ter s kožnimi/podkožnimi tumorji. Rezultati doktorske naloge so pomembni za izboljšanje protokolov in napovedovanje odgovora na zdravljenje z EKT in/ali GEP IL-12.

• Language: Slovenian
• Keywords: veterinarska onkologija, elektrokemoterapija, genski elektroprenos, bleomicin, interlevkin-12, kakovost življenja pri psih
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2020
• PID: 20.500.12556/RUL-125167
• Publication date in RUL: 05.03.2021

Secondary language

• Language: English
• Title: Evaluation of the procedures of electrochemotherapy and gene electrotransfer of plasmid DNA encoding canine interleukin-12 for the treatment of spontaneous cutaneous and oral tumours in dogs

Abstract

The aim of this doctoral dissertation was to evaluate the procedures of electrochemotherapy (ECT) and gene electrotransfer (GET) of plasmid DNA encoding canine interleukin-12 (IL-12) for the treatment of spontaneous cutaneous and oral tumours in dogs. We determined the pharmacokinetic properties of bleomycin (BLM) in the blood of the treated dogs using liquid chromatography coupled with high-resolution mass spectrometry (LC-MS/MS) and calculated the pharmacokinetic parameters. In the blood of the treated dogs, we determined the lymphocyte subpopulations (CD4+ and CD8+ lymphocytes, regulatory T cells – Treg) by flow cytometry. Immunohistochemical staining was employed to evaluate the expression of programmed cell death-1 and its ligands (PD-1 and PD-L1) in the tumours of the treated dogs. The owners of the treated dogs were offered the Cancer treatment form and Owner minitest to evaluate the quality of life of the dogs after treatment with ECT and/or IL-12 GET. The results showed that BLM had a monophasic elimination curve. The mean volume of distribution (Vd) was 224.5 ± 75.02 ml/kg, clearance (CL) 7.04 ± 2.05 ml/min/kg and elimination half-life (t1/2) 22.03 ± 0.88 min. The body weight of the dogs did not affect any of the pharmacokinetic parameters. Older dogs had lower Vd; however, the age of the dogs did not affect other pharmacokinetic parameters. No significant differences in CD4+ and CD8+ lymphocyte counts were observed during treatment. At the end of the observation period, the percentage of Treg in the blood of treated dogs decreased; the change did not correlate with the treatment response. PD-1 was expressed by 95% of mast cell tumours and 100% of oral malignant melanomas, and PD-L1 was expressed by all tumours. The dogs who had tumours with higher PD-1 and PD-L1 expression had shorter mean survival times, poorer treatment response and shorter progression-free survival than those who had tumours with lower PD-1 and PD-L1 expression. The owners assessed that the quality of their dogs' lives improved after treatment. The assessment was better in dogs that were treated with noninvasive treatments, those that responded to the treatment and those with smaller and cutaneous/subcutaneous tumours. The results of this doctoral dissertation are important for protocol optimization and prediction of the response to treatment with ECT and/or IL-12 GET.

• Keywords: veterinary oncology, electrochemotherapy, gene electrotransfer, bleomycin, interleukin-12, quality of life in dogs