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Znanstveno izhodišče: Karcinom trebušne slinavke je četrti vodilni vzrok z rakom povezane smrti v zahodnem svetu. Ob postavitvi diagnoze pri približno 40 % bolnikov kirurško zdravljenje zaradi lokalno napredovale bolezni ni mogoče. Uspešnost zdravljenja s kemoterapijo in radioterapijo je pri teh bolnikih omejena, saj je preživetje kljub terapiji kratko (< 12 mesecev). Lokalne ablacijske metode bi lahko predstavljale novo možnost zdravljenja teh bolnikov. Elektrokemoterapija (ECT) je ablacijski poseg, ki temelji na lokalni uporabi reverzibilne elektroporacije. Poseg omogoča transport i. v. apliciranega kemoterapevtika, ki sicer ne prehaja celične membrane, neposredno v celico. Metoda je že uveljavljena pri zdravljenju kožnih in jetrnih tumorjev. Do sedaj varnost in učinkovitost ECT pankreasa še nista bili sistematično proučevani. Namen dela in hipoteza: Namen doktorskega dela je raziskati varnost ECT trebušne slinavke z bleomicinom na preživetvenem modelu pujsa. Naša hipoteza je bila, da ECT normalne trebušne slinavke ne povzroča klinično pomembnih lokalnih ali sistemskih neželenih učinkov in da ECT normalne trebušne slinavke povzroči nekrozo celic v ablacijskem območju. Metode: Dovoljenje za izvajanje poskusov je izdala Etična komisija za poskuse na živalih Uprave Republike Slovenije za varno hrano, veterinarstvo in varstvo rastlin (številka dovoljenja U34401-1/2017/4). Vsi postopki so bili skladni z nacionalno in evropsko zakonodajo. Raziskava je potekala na osmih (8) prašičih, ki so bili razdeljeni v dve skupini. V prvi skupini je bil poseg ECT izveden perkutano, pod kontrolo računalniške tomografije (CT), tretiran je bil rep trebušne slinavke. V drugi skupini je bil poseg ECT izveden ob kirurški laparotomiji, tretirana sta bila glava in rep trebušne slinavke. Elektroporacijo smo izvedli z uporabo dveh igelnih elektrod (z debelino 1,2 mm) s 3 cm dolgim aktivnim delom. Obe skupini smo po posegu sledili sedem dni. Za oceno morebitnih zapletov smo spremljali klinično stanje prašičev in laboratorijske parametre. Takoj po posegu in sedmi dan po posegu smo opravili preiskavo s CT. Za oceno histoloških sprememb smo prašiče sedmi dan po posegu evtanazirali in opravili patološki pregled trebušne slinavke. Rezultati: Poseg je bil uspešno opravljen pri vseh prašičih in vsi so preživeli načrtovano opazovalno obdobje. CT-preiskava in histološki pregled nista razkrila pomembnih lokalnih neželenih učinkov. Noben od prašičev ni razvil kliničnih znakov pankreatitisa. Pri dveh prašičih (št. 6 in 8) je prišlo do prehodnega povišanja koncentracije serumske amilaze in lipaze. Histološka analiza je pri teh prašičih pokazala iatrogeno poškodbo glavnega pankreatičnega voda zaradi vstavitve igelne elektrode. Histološka analiza je v ablacijskem območju pokazala prisotnost fibroze in nekroze tik ob vstavitvi igelnih elektrod ter prisotnost apoptoze v perifernih delih ablacijskega območja. Zaključek: Raziskava je pokazala, da ECT povzroči nekrozo in apoptozo v ablacijskem območju, ki pa se ne odraža v lokalnih ali sistemskih stranskih učinkih. Zaključimo lahko, da je ECT parenhima trebušne slinavke varna ablacijska metoda. Rezultati te študije bodo prispevali k nadaljnjemu razvoju ECT trebušne slinavke. Scientific background: Pancreatic cancer is the fourth leading cause of cancer-related death in the Western world. At the time of diagnosis approximately 40% of patients present with locally advanced disease , unsuitable for surgical treatment. The success of chemotherapy and radiotherapy therapy in these patients is limited, with a short survival (

Elektroporacija je pojav, pri katerem s kratkimi visokonapetostnimi električnimi pulzi spremenimo strukturo celične membrane in s tem povečamo njeno prepustnost. Z ustrezno izbiro parametrov pulzov je pojav lahko reverzibilen ali ireverzibilen. Obe vrsti elektroporacije se uporabljata v različnih medicinskih aplikacijah. Reverzibilna elektroporacija se uporablja pri elektrokemoterapiji in genski elektrotransfekciji, ireverzibilna elektroporacija pa se uporablja za ablacijo tumorjev ter ablacijo v srcu pri zdravljenju atrijske fibrilacije. Numerične metode so pomembno orodje za analizo kompleksnih dogajanj v biološkem tkivu med elektroporacijo. Številni vidiki klinične uporabe elektroporacije so še vedno neraziskani. Numerično modeliranje nam omogoča raziskovanje novih pristopov k zdravljenju, preizkušanje novih oblik elektrod ter analizo različnih kliničnih scenarijev z vidika izvedljivosti in varnosti. Uporaba numeričnih modelov tudi zmanjša število predkliničnih in kliničnih študij, potrebnih za razvoj zdravljenja na podlagi elektroporacije. Vendar je treba modele pred vključitvijo v klinični delovni proces, npr. za načrtovanje zdravljenja, potrditi s poskusi in (pred)kliničnimi študijami. Ena najpomembnejših aplikacij numeričnega modeliranja je računalniško podprto načrtovanje zdravljenja. Predpogoj za uspeh vseh zdravljenj, ki temeljijo na elektroporaciji, je popolna pokritost kliničnega ciljnega volumna z dovolj visokim električnim poljem. Razporeditev električnega polja v tkivu je odvisna predvsem od postavitve elektrod in parametrov dovedenih pulzov. Natančno določanje porazdelitve električnega polja v tkivu, zlasti pri globoko ležečih tarčah, ni enostavna naloga. Za zagotovitev uspešnega izida zdravljenja je priporočljivo načrtovanje zdravljenja, ki temelji na numeričnih modelih, prilagojenih bolniku, in optimizacija parametrov zdravljenja. Kljub tehnološkemu napredku načrtovanje zdravljenja pri terapijah z elektroporacijo še vedno ni del standardne klinične prakse glavna omejitev izhaja iz dejstva, da se načrti trenutno pripravljajo pred posegom. Razvoj načrtovanja zdravljenja v realnem času z uporabo dejanskih položajev elektrod bo omogočil nadzor parametrov zdravljenja in izida v realnem času in je ključni korak pri uvajanju računalniško podprtega načrtovanja zdravljenja v rutinsko klinično prakso. Namen disertacije je z uporabo numeričnega modeliranja in računalniško podprtega načrtovanja zdravljenja izboljšati zdravljenje globje ležečih tumorjev z elektroporacijo. V uvodnem delu je podan pregled kliničnih aplikacij elektroporacije, sledijo osnove načrtovanja zdravljenja, ter pregled metod, ki se uporabljajo pri modeliranju elektroporacije. Glavnino disertacije sestavlja šest izvirnih znanstvenih člankov, objavljenih v mednarodnih revijah, ki zajemajo delo, opravljeno v disertaciji. Metodologija in rezultati so v člankih podrobno obravnavani, zato so na koncu disertacije povzete le razprave in zaključki posameznih člankov. V disertacijo so vključeni trije izvirni znanstveni prispevki. Prvi prispevek obsega validacijo numeričnega modela elektroporacije v jetrih z uporabo kliničnih in predkliničnih podatkov. Drugi prispevek obsega vidike varnosti in učinkovitosti zdravljenja z elektroporacijo v tveganih kliničnih scenarijih, na primer v bližini srčnih spodbujevalnikov ali ob prisotnosti kovinskih kirurških sponk v območju zdravljenja. Numerično sta bila ovrednoteni tudi izvedljivost in varnost novega pristopa k zdravljenju metastaz v hrbtenici, kar dokazuje uporabnost numeričnega modeliranja pri razvoju novih načinov zdravljenja. Tretji prispevek zajema razvoj optimizacijskega algoritma za postavitev elektrod brez uporabe računsko zahtevnih metod. Algoritem bistveno skrajša čas in strokovno znanje, ki sta potrebna za razvoj načrta zdravljenja, in predstavlja prvi korak k načrtovanju zdravljenja v realnem času. Electroporation is a phenomenon in which short high-voltage electric pulses are used to change the structural integrity of the cell membrane, thereby increasing membrane permeability. With appropriate choice of pulse parameters, the phenomenon can be reversible or irreversible. Both types of electroporation are used in various medical applications. Reversible electroporation is used in electrochemotherapy and gene electrotransfer, while irreversible electroporation is used for ablation of tumors and ablation in the heart to treat atrial fibrillation. Numerical methods are an important tool for analyzing complex interactions in biological tissue during electroporation. Many aspects of the clinical applications of electroporation are still unresolved. Numerical modeling allows us to investigate new treatment approaches, test new electrode designs, and analyze different clinical scenarios for the feasibility and safety aspects. The use of numerical models also reduces the number of preclinical and clinical studies required to develop treatments based on electroporation. However, before the models can be integrated into the clinical workflow, e.g., for treatment planning, they need to be validated by experiments and (pre)clinical studies. One of the most important applications of numerical modeling is computer-assisted treatment planning. A prerequisite for the success of all electroporation-based treatments is the complete coverage of the clinical target volume with a sufficiently high electric field. The distribution of the electric field in the tissue depends mainly on the electrode configuration and the parameters of the delivered pulse. Accurately determining the distribution of the electric field in the tissue, especially for deep-seated targets, is not a trivial task. Treatment planning based on patient-specific numerical models and optimization of treatment parameters, is advisable to ensure a successful treatment outcome. Despite the technological advances, treatment planning is still not part of the standard clinical practice for electroporation-based treatments the major limitation stems from the fact that plans are currently generated prior to the procedure. The development of real-time treatment planning using actual electrode positions will allow real-time control of treatment parameters and outcome, and is a critical step toward introducing computer-assisted treatment planning into routine clinical practice. The aim of this dissertation is to improve future electroporation-based treatments through numerical modeling and computer-assisted treatment planning. First, a brief introduction to the clinical application of electroporation is given then, the fundamentals of treatment planning are described, followed by an overview of the numerical approaches used in modeling the electroporation phenomenon. The main body of the dissertation consists of six original research papers published in international journals that comprise the work performed in the dissertation. The methodology and results are discussed in detail in the papers, therefore only the discussion and conclusions of the presented papers are reproduced at the end of the dissertation. Three original contributions to science are included within this dissertation. First, the numerical model of electroporation in the liver was validated using preclinical and clinical data. Second, the safety and efficacy aspects of electroporation-based treatments were numerically evaluated in risky clinical scenarios, such as near implanted pacemakers or in the presence of multiple metallic implants within the treatment zone. The feasibility and safety of a new treatment approach for spinal metastases was also numerically evaluated, demonstrating the utility of numerical modeling in the development of new treatments. Finally, an optimization algorithm for electrode placement was developed without using computationally intensive methods. The algorithm significantly reduces the time and expertise required to develop a treatment plan and is a step toward real-time treatment planning.

Objective: Pelvic exenteration in women with recurrent vulvar carcinoma is associated with high morbidity and mortality and substantial treatment costs. Because pelvic exenteration severely affects the quality of life and can lead to significant complications, other treatment modalities, such as electrochemotherapy, have been proposed. The aim of this study was to evaluate the feasibility and suitability of electrochemotherapy in the treatment of recurrent vulvar cancer. We aimed to analyze the treatment options, treatment outcomes, and complications in patients with recurrent vulvar cancer of the perineum. Methods: A retrospective analysis of patients who had undergone pelvic exenteration for vulvar cancer at the Institute of Oncology Ljubljana over a 16-year period was performed. As an experimental, less mutilating treatment, electrochemotherapy was performed on one patient with recurrent vulvar cancer involving the perineum. Comparative data analysis was performed between the group with pelvic exenteration and the patient with electrochemotherapy, comparing hospital stay, disease recurrence after treatment, survival after treatment in months, and quality of life after treatment. Results: We observed recurrence of disease in 2 patients with initial FIGO stage IIIC disease 3 months and 32 months after pelvic exenteration, and they died of the disease 15 and 38 months after pelvic exenteration. Two patients with FIGO stage IB were alive at 74 and 88 months after pelvic exenteration. One patient with initial FIGO stage IIIC was alive 12 months after treatment with electrochemotherapy with no visible signs of disease progression in the vulvar region, and the lesions had a complete response. The patient treated with electrochemotherapy was hospitalized for 4 days compared with the patients with pelvic exenteration, in whom the average hospital stay was 19.75 (± 1.68) days. Conclusion: Our experience has shown that electrochemotherapy might be a less radical alternative to pelvic exenteration, especially for patients with initially higher FIGO stages.

The aim of this study was to compare standard eight 100 µs pulses with alternative pulse waveforms (namely nanosecond and short high-frequency biphasic (H-FIRE) pulses) at different levels important for electroporation-based applications (extent of electrochemical reactions, time required for cell membrane resealing, decrease of cell survival and intracellular accumulation of chemotherapeutic agent after electrochemotherapy). By measuring the release of metal ions from the electrodes after electroporation, we confirmed the hypothesis that the use of H-FIRE and nanosecond pulses reduces electrochemical reactions even when the parameters of the electric field (amplitude, pause between pulses, ...) are adjusted to have the same biological effect as with longer monophasic pulses. Our second hypothesis, that electroporation of cells with different pulse shapes leading to comparable permeabilization of the cell membrane results in comparable oxidation of cellular components, could neither be confirmed nor refuted because it was shown that Click-iT Lipid Peroxidation Imaging Kit - Alexa Fluor 488 is not a suitable method for measuring lipid peroxidation in the first hours after electroporation. Our results show that nanosecond pulses with appropriately chosen parameters are suitable for use in electrochemotherapy with cisplatin and bleomycin, as they cause the same decrease in cell survival as with the standard eight 100 μs pulses. We confirmed the hypothesis that electroporation with nanosecond pulses increases the amount of chemotherapeutic agent in the cell and consequently increases its cytotoxic effect. Cilj študije je bil primerjati standardnih osem 100 µs pulzov z alternativnimi oblikami pulzov (in sicer nanosekundnimi in kratkimi visokofrekvenčnimi bipolarnimi (H-FIRE) pulzi) na različnih ravneh pomembnih za aplikacije, ki temeljijo na elektroporaciji (obseg elektrokemijskih reakcij, čas za zaceljenje celične membrane, zmanjšanje preživetja celic in znotrajcelično kopičenje kemoterapevtika po elektrokemoterapiji). Z merjenjem sproščanja kovinskih ionov z elektrod po elektroporaciji smo potrdili hipotezo, da uporaba H-FIRE in nanosekundnih pulzov zmanjšuje elektrokemijske reakcije tudi, ko so parametri električnega polja (amplituda, pavza med pulzi, …) prilagojeni tako, da imajo enak biološki učinek kot daljši monopolarni pulzi. Naše druge hipoteze, da elektroporacija celic z različnimi oblikami pulzov, ki vodijo v primerljivo permeabilizacijo celične membrane, povzroči primerljivo oksidacijo celičnih komponent, nismo mogli niti potrditi niti ovreči, ker se je izkazalo, da Click-iT Lipid Peroxidation Imaging Kit - Alexa Fluor 488 ni ustrezna metoda za merjenje lipidne peroksidacije v prvih urah po elektroporaciji. Naši rezultati kažejo, da so nanosekundni pulzi s primerno izbranimi parametri primerni za uporabo v elektrokemoterapiji s cisplatinom in bleomicinom, saj povzročijo enako zmanjšanje celičnega preživetja kot z uporabo standardnih osmih 100 μs pulzov. Potrdili smo hipotezo, da elektroporacija z nanosekundnimi pulzi poveča količino kemoterapevtika v celici in posledično poveča tudi njegov citotoksični učinek.

Nanosecond electric pulses have several potential advantages in electroporation-based procedures over the conventional micro- and millisecond pulses including low level of heating, reduced electrochemical reactions and reduced muscle contractions making them alluring for use in biomedicine and food industry. The aim of this study was to evaluate if nanosecond pulses can enhance the cytotoxicity of chemotherapeutics bleomycin and cisplatin in vitro and to quantify metal release from electrodes in comparison to 100 μs pulses commonly used in electrochemotherapy. The effects of nanosecond pulse parameters (voltage, pulse duration, number of pulses) on cell membrane permeabilization, resealing and on cell survival after electroporation only and after electrochemotherapy with bleomycin and cisplatin were evaluated on Chinese hamster ovary cells. Application of permeabilizing nanosecond pulses in combination with chemotherapeutics resulted in successful cell kill. Higher extracellular concentrations of bleomycin – but not cisplatin – were needed to achieve the same decrease in cell survival with nanosecond pulses as with eight 100 μs pulses, however, the tested bleomycin concentrations were still considerably lower compared to doses used in clinical practice. Decreasing the pulse duration from microseconds to nanoseconds and concomitantly increasing the amplitude to achieve the same biological effect resulted in reduced release of aluminum ions from electroporation cuvettes.

Kalcijeva elektroporacija (CaEP) je novejša metoda lokalnega zdravljenja tumorjev, pri kateri se za sprožitev celične smrti uporablja visoke koncentracije kalcija in elektroporacija kot dostavna metoda. Učinkovitost CaEP je že bila preizkušena v kliničnih študijah, vendar še niso pojasnjeni mehanizmi protitumorskega delovanja. V doktorski nalogi smo raziskali protitumorsko delovanje CaEP na treh nivojih. Raziskali smo direkten citotoksičen učinek na endotelijske in tumorske celice ter učinek na citoskelet in celične stike endotelijskih celic. Poleg tega smo in vivo pokazali, da ima CaEP žilno razdiralni učinek tako na normalno kot tudi tumorsko žilje, slednje pa se je izkazalo za bolj občutljivo na terapijo s CaEP. Zanimal nas je tudi imunološki učinek terapije. Zato smo preučili, ali po CaEP pride do imunogene celične smrti in sproščanja signalov nevarnosti. Pokazali smo, da se po CaEP sprošča HMGB1 in v manjši meri ATP, prav tako pride v in vitro pogojih do prerazporeditve HSP70, v in vivo pogojih pa tudi do ko-asociacije kalretikulina z membrano. Ovrednotili smo tudi uspešnost terapije na dveh različnih tumorskih modelih, mišjem melanomu B16-F10 in mišjem karcinomu 4T1, pri katerih smo v in vitro pogojih dokazali dozno odvisno preživetje tumorskih celic, prav tako imata obe celični liniji primerljivo intrinzično občutljivost na CaEP. V in vivo poskusih na podkožnih tumorjih je občutljivost tumorskih modelov zelo različna. Po terapiji z različnimi koncentracijami kalcijevih raztopin se nakazuje trend dozne odvisnosti, ki pa je nismo statististično potrdili, prav tako se je tumorski model 4T1 izkazal za bolj občutljivega na CaEP pri višjih koncentracijah. Ovrednotili smo tudi uspešnost kombinacije CaEP z genskim elektroprenosom plazmida z zapisom za IL-12, s katero lahko dodatno spodbudimo imunski odziv. Pokazali smo, da je uspešnost kombinirane terapije odvisna ne le od tumorskega modela, pač pa tudi od uspešnosti same CaEP. In sicer, znatno povečanje preživetja miši po kombinirani terapiji smo dosegli pri tumorskem modelu B16-F10, medtem ko pri modelu 4T1 dosežemo popolne odgovore že pri samem injiciranju 250 mM kalcijeve raztopine in s kombinirano terapijo ne doprinesemo k izboljšanju izida terapije. Učinkovitost terapije smo primerjali z elektrokemoterapijo z bleomicinom. Z rezultati doktorske naloge bomo pomembno prispevali k izboljšanju razumevanja delovanja CaEP. Calcium electroporation (CaEP) is a novel local ablative method for tumor treatment, where high concentrations of calcium are used for induction of cell death, and electroporation is used as a delivery method. The effectiveness of CaEP was already tested in several clinical studies however, its mechanisms are not yet completely clarified. In this study, the effectiveness of CaEP was studied on three levels. First, the direct cytotoxic effect on endothelial and tumor cell lines was tested, as well as the effects on cytoskeleton and cell-cell junctions of endothelial cells. In in vivo studies was shown, CaEP has a vessel disrupting effect, where tumor vasculature was shown to be more sensitive to CaEP than normal. Besides that, we tested immunological consequences of CaEP on two different tumor models, murine melanoma B16-F10 and murine carcinoma 4T1. We showed in B16-F10 tumor model that induction of immunogenic cell death occurs after CaEP, as excretion of HMGB1 and ATP transpires, as well as translocation of HSP70 to the cell surface, in ex vivo experiments, co-association of calreticulin with membranes was confirmed. We also evaluated the effectiveness of different concentrations of calcium, where we showed dose-dependent survival of cells with similar sensitivity of both cell lines to CaEP however, in subcutaneous tumors in vivo, this was not the case. Here, 4T1 tumor model proved to be more sensitive to CaEP as B16-F10. A combination of CaEP with gene electrotransfer of plasmid encoding IL-12 was also evaluated, which proved to be dependent on tumor type as well as on the effectiveness of CaEP alone. Combined therapy showed significantly enhanced survival in B16-F10 model, whereas, in 4T1 model, calcium injection alone resulted in tumor cures and IL-12 did not improve the outcome. Electrochemotherapy was used as a positive control in all experiments. The results of this study will importantly contribute to the understanding of CaEP mechanisms.

Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4%T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status. Bibliografija na koncu prispevka. Abstract.

Objectives: The use of thermal ablative therapies in the pancreatic tumors is limited because of the risk of the vessel injury and potential pancreatitis or fistula formation. Electrochemotherapy (ECT) is an ablative therapy with established role in the treatment of cutaneous and liver tumors. This study was designed to evaluate the safety and feasibility of ECT of the pancreas in a porcine survival model. Methods: In the first group, 4 animals underwent computed tomography (CT)%guided percutaneous ECT with bleomycin of the pancreatic tail. In the second group (4 animals), the intraoperative ECTwith bleomycin of pancreatic tail and head was performed. Animals were followed for 7 days and then killed. Clinical parameters, CT imaging, laboratory, and histologic analysis were performed. Results: All pigs survived the ECT procedure and none of them developed clinical signs of acute pancreatitis or related complications. There were no signs of acute pancreatitis or damage to the large vessels present in the follow-up CT scans. No significant change in laboratory parameters was obtained after procedure. Conclusions: This study shows that ECT with bleomycin is feasible and safe in the pancreatic parenchyma. Clinical studies are needed to evaluate the efficacy of ECT in pancreatic cancer.

Currently, in high-frequency electroporation, much progress has been made but limited to research groups with custom-made laboratory prototype electroporators. According to the review of electroporators and economic evaluations, there is still an area of pulse parameters that needs to be investigated. The development of an asymmetric bipolar pulse generator with a maximum voltage of 4 kV and minimum duration time of a few hundred nanoseconds, would enable in vivo evaluation of biological effects of high-frequency electroporation pulses. Herein, from a series of most commonly used drivers and optical isolations in high-voltage pulse generators the one with optimal characteristics was used. In addition, the circuit topology of the developed device is described in detail. The developed device is able to generate 4 kV pulses, with theoretical 131 A maximal current and 200 ns minimal pulse duration, the maximal pulse repetition rate is 2 MHz and the burst maximal repetition rate is 1 MHz. The device was tested in vivo. The effectiveness of electrochemotherapy of high-frequency electroporation pulses is compared to “classical” electrochemotherapy pulses. In vivo electrochemotherapy with high-frequency electroporation pulses was at least as effective as with “classical” well-established electric pulses, resulting in 86% and 50% complete responses, respectively. In contrast to previous reports, however, muscle contractions were comparable between the two protocols.

Elektrokemoterapija je način zdravljenja, ki uporablja elektroporacijo v kombinaciji s citostatikom, bodisi bleomicinom ali cisplatinom, za dosego lokalne kontrole tumorja. Bleomicin v celico vstopa s prenašalnimi proteini po endocitotski poti, cisplatin pa s pomočjo bakrovih kanalčkov in pasivno difuzijo. Ker je nabor prenašalnih proteinov precej majhen, je posledično tudi privzem citostatika v celico omejen. Elektrokemoterapija temelji na električnih pulzih, ki destabilizirajo celično membrano v tumorjih in tako se bistveno poveča vnos hidrofilnih citostatikov, ki imajo oviran transport skozi celično membrano. Bleomicin je protitumorsko zdravilo, ki prek tvorbe dvojnih prelomov ustavi celično delitev in povzroča smrt celic. Cisplatin pa uničujejo rakave celice tako, da poškodujejo DNA tako da z DNA tvori adukte, zavira sintezo in podvojevanje DNA in povzroči apoptotično smrt celic. Glavni cilj magistrske naloge je bil določiti občutljivost celic raka trebušne slinavke na elektrokemoterapijo s cisplatinom ali bleomicinom. Zanimalo nas je tudi, ali se citotoksični učinek poveča, če tretiramo celice raka trebušne slinavke s kombinacijo elektrokemoterapije s citostatikom in sunitinibom, saj tega do sedaj še nismo zasledili v literaturi. Sunitinib malat je molekula oksindola, zasnovana za selektivno interakcijo z znotrajceličnimi mesti ATP receptorjev kinaz kot so VEGFR1–3, PDGFR, KIT, FLT3 in CSF1R. Citotoksično delovanje kombinirane terapije smo preverili na humani celični liniji BxPC-3 raka trebušne slinavke. S testom PrestoBlueTM smo določili podvojitveni čas celične linije BxPC-3, ki je 48 ur (2 dni). Najbolj optimalno so rastle celice, ko smo jih nasadili 2000 / vdolbinico. Dokazali smo, da so celice BxPC-3 bolj občutljive na elektrokemoterapijo z bleomicinom kot na elektrokemoterapijo s cisplatinom. Kombinacija elektrokemoterapije z inhibitorjem tirozin kinaz sunitinibom na rakavih celicah trebušne slinavke, je imela večji citotoksični učinek le v primeru uporabe bleomicna. Kombinirana terapija elektrokemoterapije z 0,14 µM bleomicinom in sunitinibom v treh različnih koncentracijah (2,5, 5 in 7,5 µM) je imela sinergistični učinek. Glede na pozitivne in vitro rezultete, bi bilo smiselno tovrstno zdravljenje testirati tudi na tumorskem modelu trebušne slinavke in vivo. Electrochemotherapy is a treatment option, using electroporation in combination with cytotoxic drugs, either bleomycin or cisplatin, to achieve local tumor control. Bleomycin enters the cell through endocytic transfer proteins and cisplatin through copper channels and passive diffusion. Because the set of transport proteins is quite small, consequently the uptake of the cytostatic into the cell is also limited.The set of transporter proteins is quite small, therefore the uptake of cytostatics into the cell is limited. Electrochemotherapy is based on electrical pulses that destabilize the cell membrane in tumors and thus significantly increase the cellular uptake of hydrophilic cytostatics that have obstructed transport across the cell membrane. Bleomycin is an antitumor drug that stops cell division through the formation of DNA double strand breaks leading to cell death. Cisplatin, on the other hand, destroy cancer cells by damaging DNA with formation of DNA adducts, consequently inhibiting DNA synthesis and replication, and causing apoptotic cell death. The aim of the master's thesis was to determine the sensitivity of pancreatic cancer cells to electrochemotherapy with cisplatin or bleomycin. We also investigated whether the cytotoxic effect is enhanced when pancreatic cancer cells are treated with combined electrochemotherapy and sunitinib, as this has not been described in the literature to date. Sunitinib malate is an oxindole molecule known to selectively interact with intracellular sites of ATP receptor kinases such as VEGFR1–3, PDGFR, KIT, FLT3, and CSF1R. The cytotoxic effect of combined therapy was examined on the human BxPC-3 pancreatic cancer cell line. The PrestoBlueTM assay was used to determine the doubling time of the BxPC-3 cell line, which was equal to 48 hours (2 days). Most optimal cell growth was observed when we plated 2000 cells per well. We demonstrated that BxPC-3 cells were more sensitive to electrochemotherapy with bleomycin than with cisplatin. Combination of electrochemotherapy with the tyrosine kinase inhibitor sunitinib on pancreatic cancer cells had more pronounced cytotoxic effect than electrochemotherapy alone, only when bleomycin was used. Combination therapy of electrochemotherapy with 0.14 µM bleomycin and sunitinib in three different concentrations (2.5, 5 in 7.5 µM) had a synergistic effect. Given the positive in vitro results, it would make sense to test this type of treatment on a tumor model of the pancreas in vivo.

Namen doktorske disertacije je bil ovrednotenje rezultatov slikovno-diagnostičnih metod po elektroporaciji (EP) in elektrokemoterapiji (EKT) jeter zdravih prašičev za preučitev varnostnega vidika EKT globoko ležečih jetrnih tumorjev ter rezultatov ultrazvočnega pregleda s kontrastnim sredstvom (UZ KS) po EKT in genskem elektroprenosu (GEP) mišjih in pasjih tumorjev kot prediktivnih dejavnikov zdravljenja. Slikovno-diagnostične spremembe po EP in EKT z bleomicinom (BLM) v jetrnem parenhimu in velikih krvnih žilah jeter zdravega prašiča s potencialno nevarno vstavitvijo linearnih in heksagonalnih elektrod v velike jetrne žile smo preučili z ultrazvočno preiskavo takoj po EP/EKT ter z računalniško tomografijo od 60 do 90 minut in en teden po EP/EKT. Rezultati slikovno-diagnostičnih metod jeter prašičev po EK so pokazali zmanjšano perfuzijo tretiranega področja. Spremembe niso bile posledica uporabe BLM, saj so bile enake pri poskusni in kontrolni skupini, kjer smo aplicirali samo električne pulze. Namerna vstavitev elektrod in aplikacija električnih pulzov neposredno v svetlino velikih žil ni povzročila tromboz, krvavitev ali drugih hemodinamskih motenj. Kot model preiskovanja uporabnosti UZ-KS kot prediktivnega dejavnika zdravljenja s terapevtskimi metodami, ki temeljijo na EP, smo uporabili miši z induciranimi melanomi. V prvi raziskavi smo na mišjem modelu, ki smo ga zdravili z obsevanjem in GEP plazmidne DNA, ki utiša adhezijsko molekulo celic melanoma, opazili značilno zmanjšanje perfuzije tumorjev v terapevtskih skupinah v primerjavi s kontrolnimi skupinami, rezultati UZ-KS pa so korelirali z zmanjšano gostoto žil. Povprečne vrednosti parametra, ki opisuje volumen pretoka, so bile značilno nižje pri tumorjih, ki so se odzvali na terapijo s popolnim odgovorom, in vrednosti PE so pokazale trend korelacije s protitumorsko učinkovitostjo. V drugi raziskavi na mišjih melanomih, zdravljenih z EKT z BLM v kombinaciji z GEP plazmida za interlevkin 12 (pIL-12), je UZ-KS pokazal zmanjšano perfuzijo v skupinah z daljšim časom podvojitve volumna tumorja in prediktivno vrednost UZ KS smo potrdili s statistično značilnimi Pearsonovimi koeficienti. S krajšim časom podvojitve volumna tumorja je bila povezana tudi večja heterogenost perfuzije tumorja. V klinični del raziskave smo vključili pse s kožnimi in podkožnimi tumorji, ki so bili zdravljeni s standardnim postopkom kombinacije EKT in GEP pIL-12. Rezultate UZ-KS smo primerjali z izidom zdravljenja. Tudi v klinični raziskavi pri psih smo ugotovili številne razlike v parametrih UZ KS med tumorji s popolnim odgovorom in tumorji z nepopolnim odgovorom: perfuzija in heterogenost perfuzije sta bili manjši pri tumorjih s popolnim odgovorom. The aim of this dissertation was to evaluate the results of diagnostic imaging after electroporation (EP) and electrochemotherapy (ECT) of porcine liver to investigate the safety aspect of ECT of deep-seated liver tumors and the results of dynamic contrast-enhanced ultrasound (DCE US) after ECT and gene electrotransfer (GET) of murine and canine tumors as potential predictive factors in treatment. Radiological findings after EP and ECT with bleomycin (BLM) on the hepatic parenchyma and major blood vessels of the liver of a healthy pig with potentially dangerous insertion of the electrodes into the major hepatic vessels were examined by ultrasound immediately after ECT and by computed tomography from 60 to 90 minutes and one week after ECT with linear and hexagonal electrodes. The results of diagnostic imaging of the porcine liver after ECT showed decreased blood flow in the treated area. The changes were not due to the use of BLM, as they were the same in the experimental and control groups, where only electrical pulses were administered. Intentional insertion of electrodes and application of electrical pulses directly into the lumen of large vessels did not result in thrombosis, haemorrhage, or other hemodynamically significant injury. Mice with induced melanoma were used as a model to investigate the utility of DCE-US as a prognostic factor for treatment with EP-based therapies. In the first study in a mouse model treated with irradiation and GET of plasmid DNA that silences the melanoma cell adhesion molecule, we observed a decrease in tumor perfusion in the treatment groups compared with the control groups, and the results of DCE US correlated with decreased vessel density detected histologically. Furthermore, mean values of parameters describing flow volume were significantly lower in tumors with complete response, and they showed a trend toward correlation with antitumor efficacy. In the second study, mouse melanomas were treated with ECT in combination with GET of plasmid DNA encoding interleukin-12 (pIL-12). DCE US showed reduced perfusion in groups with longer tumor volume doubling time, and the predicted DCE US value was confirmed by statistically significant Pearson coefficients. In addition, greater tumor perfusion heterogeneity was associated with shorter tumor doubling time. The clinical study included dogs with cutaneous and subcutaneous neoplasms treated with a standard procedure combining ECT and GET pIL-12. We also found a number of differences in DCE-US parameters between tumors with complete response and tumors with incomplete response in the clinical study: Perfusion and perfusion heterogeneity were lower in tumors with complete response.

Elektroporacija je fizikalna metoda, ki omogoča prehajanje številnih molekul skozi celično membrano, ki jih celica v normalnih pogojih ne prepušča. Z uporabo kratkih, visokonapetostnih električnih pulzov v plazemski membrani povzročimo nastanek hidrofilnih por. Ločimo reverzibilno elektroporacijo, po kateri se celična membrana zaceli in ireverzibilno elektroporacijo, po kateri se celična membrana ne more regenerirati ter posledično pride do celične smrti. Elektroporacija ima veliko možnih aplikacij na več področjih. Uporablja se jo za zlivanje celic, transfekcijo ali transformacijo celic, sterilizacijo vode, pasterizacijo hrane, ekstrakcijo snovi. Elektroporacija je dobila tudi mesto v medicini, na področju zdravljenja raka. Za ta namen lahko uporabimo reverzibilno elektroporacijo, s katero omogočimo, da v rakaste celice prodrejo citostatiki in tako povečamo njihov učinek. Pri uporabi citostatika cisplatin z elektroporacijo povečamo njegovo učinkovitost za 70-krat, pri bleomicinu pa kar do 700-krat. Možna je tudi uporaba ireverzibilne elektroporacije, kjer rakaste celice ubijemo in jo uporabljamo za netermično ablacijo tkiv in tumorjev. Electroporation is a physical method that allows the passage of many molecules through the cell membrane that is impermeable to this very molecules. Using short, high-voltage electrical pulses in the plasma membrane causes the formation of hydrophilic pores. Membrane electroporation can either be reversible (cell membrane reseal) or irreversible (cell membrane cannot return to its primary state and cell death occurs). Electroporation has many possible applications in several areas. It is used for cell fusion, water sterilization, food pasteurization, substance extraction, transfection, transformation, etc. Electroporation has also gained a place in medicine, especially in the field of cancer treatment. For this purpose, reversible electroporation is used to introduce cytostatic into cancer cells. When electroporation is used, the efficiency of cisplatin increases 70 times and the efficiency of bleomycin up to 700 times. We can also use irreversible electroporation, where we kill cancer cells, for non-thermal ablation of tissues and tumors.

Electrochemotherapy (ECT) is an effective locoregional therapy for cutaneous melanoma metastases and has been safely combined with immune checkpoint inhibitors in preliminary experiences. Since ECT is known to induce immunogenic cell death, its combination with immune checkpoint inhibitors might be beneficial. In this study, we aimed to investigate the effectiveness of ECT on cutaneous melanoma metastases in combination with pembrolizumab. We undertook a retrospective matched cohort analysis of stage IIIC%IV melanoma patients, included in the International Network for sharing practices of ECT (InspECT) and the Slovenian Cancer Registry. We compared the outcome of patients who received the following treatments: (a) pembrolizumab alone, (b) pembrolizumab plus ECT, and (c) ECT. The groups were matched for age, sex, performance status, and size of skin metastases. The local objective response rate (ORR) was higher in the pembrolizumab-ECT group than in the pembrolizumab group (78% and 39%, p < 0.001). The 1 year local progression-free survival (LPFS) rates were 86% and 51% (p < 0.001), and the 1 year systemic PFS rates were 64% and 39%, respectively (p = 0.034). The 1 year overall survival (OS) rates were 88% and 64%, respectively (p = 0.006). Our results suggest that skin-directed therapy with ECT improves superficial tumor control in melanoma patients treated with pembrolizumab. Interestingly, we observed longer PFS and OS in the pembrolizumab-ECT group than in the pembrolizumab group. These findings warrant prospective confirmation. Nasl. z nasl. zaslona. Opis vira z dne 31. 8. 2021. Bibliografija na koncu prispevka. Abstract.

The plasma concentration profile of bleomycin in the distribution phase of patients younger than 65 years is needed to determine the suitable time interval for efficient application of electric pulses during electrochemotherapy. Additionally, bleomycin concentrations in the treated tumors for effective tumor response are not known. In this study, the pharmacokinetic profile of bleomycin in the distribution phase in 12 patients younger than 65 years was determined. In 17 patients, the intratumoral bleomycin concentration was determined before the application of electric pulses. In younger patients, the pharmacokinetics of intravenously injected bleomycin demonstrated a faster plasma clearance rate than that in patients older than 65 years. This outcome might indicate that the lowering of the standard bleomycin dose of 15,000 IU/m$^2$ with intravenous bleomycin injection for electrochemotherapy is not recommended in younger patients. Based on the plasma concentration data gathered, a time interval for electrochemotherapy of 5–15 min after bleomycin injection was determined. The median bleomycin concentration in tumors 8 min after bleomycin injection, at the time of electroporation, was 170 ng/g. Based on collected data, the reduction of the bleomycin dose is not recommended in younger patients however, a shortened time interval for application of electric pulses in electrochemotherapy to 5–15 min after intravenous bleomycin injection should be considered.

Endometrial carcinoma is the most common gynecological malignancy and the fifth most common malignancy in women. The worldwide incidence is 15.9 new cases per 100,000 women per year, and the incidence in Europe is 22.7 new cases. Minority of cases are diagnosed at an advanced stage of the disease. Cutaneous metastases are very rare with a prevalence of 0.8%. If cutaneous metastases are present, the prognosis is poor with an overall survival of up to 12 months. In this review, we presented clinical data on treatment of gynecological cancers with electrochemotherapy, with focus on treatment of cutaneous vulvar metastases from endometrial cancer. Further, we present our data on the case of a 64-year-old woman with recurrent endometrial adenocarcinoma with vulvar skin metastases. Treatment of endometrial carcinoma metastases is multimodal with surgery, chemotherapy, radiotherapy and hormone treatment. There is still no consensus about the specific treatment of cutaneous metastases from endometrial cancer, in particular in order to release symptoms. Electrochemotherapy may be a treatment option to reduce pain and bleeding and a safe option to treat multiple skin metastases.

Electrochemotherapy (ECT) is a local ablative treatment that is based on the reversible electroporation and intracellular accumulation of hydrophilic drug molecules, which greatly increases their cytotoxicity. In mucosal head and neck cancer (HNC), experience with ECT is limited due to the poor accessibility of tumors. In order to review the experience with ECT in mucosal HNC, we undertook a systematic review of the literature. In 22 articles, published between 1998 and 2020, 16 studies with 164 patients were described. Curative and palliative intent treatment were given to 36 (22%) and 128 patients (78%), respectively. The majority of tumors were squamous cell carcinomas (79.3%) and located in the oral cavity (62.8%). In the curative intent group, complete response after one ECT treatment was achieved in 80.5% of the patients, and in the palliative intent group, the objective (complete and partial) response rate was 73.1% (31.2% and 41.9%). No serious adverse events were reported during or soon after ECT and late effects were rare (19 events in 17 patients). The quality-of-life assessments did not show a significant deterioration at 12 months post-ECT. Provided these preliminary data are confirmed in randomized controlled trials, ECT may be an interesting treatment option in selected patients with HNC not amenable to standard local treatment. Nasl. z nasl. zaslona. Opis vira z dne 3. 5. 2021. Bibliografija na koncu prispevka. Abstract.

The beneficial effects of electrochemotherapy (ECT) for superficial tumours and, more recently, deepseated malignancies in terms of local control and quality of life are widely accepted. However, the variability in responses across histotypes needs to be explored. Currently, patient selection for ECT is based on clinical factors (tumour size, histotype, and exposure to previous oncological treatments), whereas there are no biomarkers to predict the response to treatment. In this field, two major areas of investigation can be identified, i.e., tumour cell characteristics and the tumour microenvironment (vasculature, extracellular matrix, and immune infiltrate). For each of these areas, we describe the current knowledge and discuss how to foster further investigation. This review aims to provide a summary of the currently used guiding clinical factors and delineates a research roadmap for future studies to identify putative biomarkers of response to ECT. These biomarkers may allow researchers to improve ECT practice by customising treatment parameters, manipulating the tumour and its microenvironment, and exploring novel therapeutic combinations.

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. 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.

Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast can-cer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to com-bine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin inducedDNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied inBRCA1mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines.Therapeutic effectiveness was studied in 2D and 3D cell cultures andin vivoon subcutaneousHCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined withthe evaluation ofcH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated theeffectiveness of ECT inBRCA1mutated HCC1937, but not in non-mutated HCC1143 cells. The combinedtherapy had a synergistic effect, which was due to the increased number of DNA double strand breaks.Addition of olaparib to ECT with bleomycinin vivoin HCC1937 tumor model had only minimal effect,indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repar inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin. Bibliografija na kocu poglavja. Abstract.

The aim of this clinical study was to investigate the effectiveness and long-term safety of electrochemotherapy as an emerging treatment for HCC in patients not suitable for other treatment options. A prospective phase II clinical study was conducted in patients with primary HCC who were not suitable for other treatment options according to the Barcelona Clinic Liver Cancer classification. A total of 24 patients with 32 tumors were treated by electrochemotherapy. The procedure was effective, feasible, and safe with some procedure-related side effects. The responses of the 32 treated nodules were: 84.4% complete response (CR), 12.5% partial response (PR), and 3.1% stable disease (SD). The treatment was equally effective for nodules located centrally and peripherally. Electrochemotherapy provided a durable response with local tumor control over 50 months of observation in 78.0% of nodules. The patient responses were: 79.2% CR and 16.6% PR. The median progression-free survival was 12 months (range 2.7&ndash, 50), and the overall survival over 5 years of observation was 72.0%. This prospective phase II clinical study showed that electrochemotherapy was an effective, feasible, and safe option for treating HCC in patients not suitable for other treatment options.

Elektroporacija (EP) je fizikalna metoda s katero z dovajanjem električnih pulzov v celični membrani za kratek čas ustvarimo pore, kar olajša prehod v celice določenim molekulam. Za zdravljenje rakavih obolenj se vse bolj uporablja EP v kombinaciji s cisplatinom (CDDP) in bleomicinom, čemur rečemo elektrokemoterapija (EKT). Cisplatin je platinova (II) spojina, ki s kovalentnimi povezavami povzroči spremembe v strukturi DNA in vodi v smrt rakave celice. Kljub temu, da je CDDP eden najpogosteje uporabljenih kemoterapevtikov, pa povzroča hude stranske učinke in tudi odpornost rakavih celic. Zaradi tega se raziskave usmerjajo v razvoj novih platinovih (II) analogov s protitumorskim delovanjem, kamor spadata tudi [PtCl (5,7-dibromo-8-hidroksikinolinato)(S-dmso)] (spojina A) in njen analog, [PtCl (5,7-dibromo-8-hidroksikinolinato)(pta)] (spojina B). Citotoksičnost obeh spojin v kombinaciji z EP smo preverili na humani celični liniji raka debelega črevesja (HT29) in raka dojke (MCF7). S testom klonogenosti smo izračunali vrednosti IC50 in jih primerjali z vrednostmi IC50 CDDP, s pripravo citospinov in barvanjem po Giemsi pa smo na podlagi morfoloških značilnosti ugotovili delež apoptotičnih in nekrotičnih celic. Ugotovili smo, da spojini A in B delujeta citotoksično na tumorske celice in povzročata predvsem apoptozo ter deloma nekrozo. Elektroporacija statistično značilno poveča citotoksično delovanje vseh treh platinovih (II) spojin in je učinkovita metoda za izboljšanje vnosa ne samo CDDP, pač pa tudi spojine A in B. Spojina B se je v kombinaciji z in brez EP izkazala za bolj toksično od spojine A in hkrati tudi enako ali celo bolj citotoksično kot CDDP pri obeh celičnih linijah in je zato primerna za nadaljne testiranje, kot potencialni kemoterapevtik. Electroporation (EP) is a physical technique in which an electric field is applied to cells in order to increase the permeability of cell membrane for poorly permeating molecules. Electroporation in combination with cisplatin (CDDP), also called electrochemotherapy (ECT), is used for local treatment of several malignant tumors. Cisplatin is a well-known platinum (II) antitumor drug which antitumor activity has been linked to its ability to form DNA adducts, which subsequently lead to apoptosis of cancer cells. Although CDDP is broadly used, its main limitations are severe side effects and tumor cells’ resistance. Because of this, researchers are focusing on developing new platinum (II) complexes with antitumor activity, including [PtCl (5,7-dibromo-8-hydroxyquinolinato)(S-dmso)] (compound A) and it's analog, [PtCl (5,7-dibromo-8-hydroxyquinolinato)(pta)] (compound B). In the thesis, we analysed the cytotoxicity of compound A and B in combination with EP in human colon cancer (HT29) and human breast cancer cell line (MCF7). We determined IC50 values with clonogenic assay and compared it to IC50 values of CDDP. We used Giemsa staining to determine the percentage of apoptotic and necrotic cells. Results show that all compounds, A, B and CDDP have a cytotoxic effect on both tumor cell lines and cause primarily apoptotic cell death. Furthermore, EP has proved to be a very effective method for drug uptake as it has significantly improved cytotoxicity of all three platinum (II) complexes. Compound B has turned out to be more cytotoxic with and without EP than compound A and showed similar or better cytotoxicity than CDDP in both cell lines. Based on our results, compound B is very promising potential therapeutic agent.

Electroporation is a method of inducing an increase in permeability of the cell membrane through the application of an electric field and can be used as a delivery method for introducing molecules of interest (e.g., chemotherapeutics or plasmid DNA) into cells. Electroporation-based treatments (i.e., electrochemotherapy, gene electrotransfer, and their combinations) have been shown to be safe and effective in veterinary oncology, but they are currently mostly recommended for the treatment of those solid tumors for which clients have declined surgery and/or radiotherapy. Published data show that electroporation-based treatments are also safe, simple, fast and cost-effective treatment alternatives for selected oral and maxillofacial tumors, especially small squamous cell carcinoma and malignant melanoma tumors not involving the bone in dogs. In these patients, a good local response to treatment is expected to result in increased survival time with good quality of life. Despite emerging evidence of the clinical efficacy of electroporation-based treatments for oral and maxillofacial tumors, further investigation is needed to optimize treatment protocols, improve clinical data reporting and better understand the mechanisms of patients' response to the treatment.

Rak je druga po smrtnosti najučestalija bolest na svijetu. Zbog svoje kompleksnosti, liječenje još uvijek nije efikasno u mnogim slučajevima. Zbog raznolikih vrsta tumora i mutacija koje se pojavljuju, veoma je teško pronaći standardiziranu terapiju. Korištenje električnih polja spada u novije metode pri liječenju tumora i još je relativno neistraženo, ali obećavajuće područje. Poboljšava unos lijekova u stanicu, koristi se kod genske terapije i u ablaciji tumorskog tkiva. Terapija je veoma sigurna, nuspojave nisu izražene i postotak oporavka pacijenata je veoma visok. Dva su glavna ishoda djelovanja električnog polja na membranu. Ireverzibilna i reverzibilna elektroporacija. Kod ireverzibilne elektroporacije, struktura membrane je trajno narušena i ona se neće vratiti u prvotno stanje, te se time postiže smrt stanice. Kod reverzibilne elektroporacije, membrana se vraća u prvobitno stanje, te ne dolazi do smrti stanice. Elektrokemoterapija je poboljšan način dostave lijeka u tumorsku stanicu. Pomoću električnog polja se povećava propusnost membrane, te lijek lakše ulazi u tumorsku stanicu. Primjenom elektrokemoterapije, smanjuje se potreba doza kemoterapeutike za liječenje, broj nuspojava je reduciran, profil liječenja je poboljšan. Bleomicin i cisplatin su indentificirani kao lijekovi koji bi mogli imat poboljšanu terapiju sa elektrokemoterapijom. Istraživanja sa bleomicinom su pokazala da ima 50% poboljšano djelovanje kada se primjenjuje sa elektrokemoterapijom, u odnos na primjenjivanje njega samog. Genska terapija je nov pristup liječenju tumora. Može se koristiti da se ispravi gubitak funkcije uzrokovan mutacijom ili da se eksprimira genski proizvod s nedostatkom na fiziološkoj razini. Cancer is the second most common disease in the world. Due to its complexity, treatment is still not effective in many cases. Due to the various types of tumors and mutations that occur, it is very difficult to find standardized therapy. The use of electric fields is one of the newer methods in the treatment of tumors and is still a relatively unexplored but promising area. It improves the uptake of drugs into the cell, is used in gene therapy and in the ablation of tumor tissue. The therapy is very safe, the side effects are not pronounced and the recovery rate of the patients is very high. There are two main outcomes of the action of an electric field on a membrane. Irreversible and reversible electroporation. In irreversible electroporation, the structure of the membrane is permanently disrupted and it will not return to its original state, thus achieving cell death. With reversible electroporation, the membrane returns to its original state, and cell death does not occur. Electrochemotherapy is an improved way of delivering a drug to a tumor cell. The electric field increases the permeability of the membrane, and the drug enters the tumor cell more easily. With the application of electrochemotherapy, the need for doses of chemotherapy for treatment is reduced, the number of side effects is reduced, the treatment profile is improved. Bleomycin and cisplatin have been identified as drugs that could have improved electrochemotherapy. Studies with bleomycin have shown that it has a 50% improved effect when administered with electrochemotherapy, compared to self-administration. Gene therapy is a new approach to treating tumors. It can be used to correct a loss of function caused by a mutation or to express a deficient gene product at the physiological level.

Izhodišča: Elektrokemoterapija je učinkovita lokalna ablativna metoda za zdravljenje različnih vrst tumorjev, pri kateri se uporablja intratumorski ali intravenski vnos kemoterapevtikov (bleomicina ali cisplatina) v kombinaciji z elektroporacijo. Na učinkovitost elektrokemoterapije vplivajo tako pravilnost izvedbe terapije, kot tudi lastnosti samih tumorjev. Med drugim so v kliničnih študijah opažali slabši odgovor tumorjev, ki so bili predhodno že zdravljeni s kemo- ali radioterapijo. Eden izmed možnih vzrokov bi lahko bila pridobljena intrinzična rezistenca tumorskih celic na kemoterapevtike. Recidivni tumorji namreč izrastejo iz najbolj odpornih celic, ki so zaradi določenih prilagoditev, kot je boljše popravilo poškodb DNA, preživele predhodno obsevalno terapijo. Manj je znano, kako te spremembe vplivajo na kemorezistenco celic in posledično na rezistenco na elektrokemoterapijo. Namen: V doktorski nalogi smo se namenili raziskati mehanizme intrinzične kemo- in radiorezistence recidivnih tumorjev po predhodnem obsevanju, ki lahko vplivajo na učinkovitost elektrokemoterapije. Hipoteze: Preverjali smo dve hipotezi. (1) Slabša občutljivost radiorezistentnih tumorskih celic na elektrokemoterapijo je pogojena tudi z intrinzično rezistenco na cisplatin in bleomicin in (2) Razvoj kemorezistence pri radiorezistentnih tumorskih celicah je pogojen tako s spremenjenimi mehanizmi popravila poškodb DNA, kot tudi s spremenjenim celičnim privzemom kemoterapevtikov. Materiali in metode: V naših poskusih smo primerjali dve izogeni humani celični liniji ploščatoceličnega karcinoma žrela: starševsko (FaDu) in radiorezistetno podlinijo (FaDu-RR), ki smo jo vzpostavili s frakcioniranim obsevanjem linije FaDu. S testom klonogenosti smo najprej določili stopnjo občutljivosti obeh celičnih linij na obsevanje, kontinuirano izpostavljenost cisplatinu ali bleomicinu, ter na elektrokemoterapijo. Za izvedbo in vivo poskusov smo s podkožnim injiciranjem ene ali druge celične linije na bok imunokompromitiranih (SCID) miši inducirali FaDu in FaDu-RR tumorje. Tumorska modela smo primerjali glede nekaterih histoloških značilnosti ter občutljivosti na obsevanje, kemoterapijo s cisplatinom, kombinacijo obsevanja in cisplatina ter elektrokemoterapijo. Učinek naštetih terapij smo spremljali z merjenjem zaostanka v rasti tumorjev v primerjavi s kontrolno skupino ter z izrisom krivulj preživetja. V drugem delu naloge smo celični liniji po izpostavitvi cisplatinu ali bleomicinu primerjali glede sposobnosti popravila dvojnih prelomov DNA (z imunocitokemičnim določanjem žarišč ?-H2AX) ter glede na izraženost genov za popravilo poškodb DNA (z metodo PCR). Z visoko specifičnimi analičnimi metodami, ki temeljijo na masni spektrometriji, smo primerjali tumorski privzem cisplatina in bleomicina po kemo- in elektrokemoterapiji. Rezultati: S frakcioniranim obsevanjem smo uspešno vzpostavili radiorezistentno celično linijo FaDu-RR z induciranimi obrambnimi mehanizmi, ki so poleg pridobljene radiorezistence vodili tudi do navzkrižne rezistence na cisplatin. Dodatno smo v in vivo pogojih dokazali večjo odpornost FaDu-RR tumorjev na kombinacijo frakcioniranega obsevanja in cisplatina ter na elektrokemoterapijo s cisplatinom. Pomemben rezultat te naloge je tudi, da je elektrokemoterapija z bleomicinom pri obeh celičnih linijah oziroma tumorskih modelih enako učinkovita. V drugem delu naloge, kjer smo raziskovali morebitne mehanizme rezistence, smo dokazali, da radiorezistentne celice poleg zmanjšanega intracelularnega kopičenja cisplatina (v primerjavi s starševsko celično linijo) učinkoviteje popravljajo določene tipe poškodb DNA ter imajo spremenjen spekter izražanja genov za popravilo poškodb DNA. Zaključki: Frakcionirano obsevanje je v radiorezistetni celični liniji sprožilo nekatere mehanizme prilagoditve, kot sta bolj učinkovito popravilo poškodb DNA in navzkrižna rezistenca na cisplatin. V in vivo pogojih so bili FaDu-RR tumorji v primerjavi s FaDu tumorji enako občutljivi na elektrokemoterapijo na osnovi bleomicina, ter bolj odporni na elektrokemoterapijo na osnovi cisplatina. Glede na dejstvo, da je bila ugotovljena razlika med tumorskima modeloma majhna, na osnovi teh rezultatov lahko izključimo pomembnejšo vlogo intrinzične radiorezistence pri slabšem odzivu predhodno obsevanih tumorjev na elektrokemoterapijo. Zaradi navzkrižne rezistence radiorezistentnih celic na cisplatin, bi pri zdravljenju takih tumorjev priporočili elektrokemoterapijo na osnovi bleomicina. Background: Electrochemotherapy is an effective local ablative method for treatment of different types of tumours, where intratumoral or intravenous chemotherapeutic drug administrantion (cisplatin or bleomycin) is combined with electroporation. Effectiveness of this method depends on technical performance and on tumor characteristics. It was suggested in clinical studies that the pre-treatment with radio- or chemotherapy significantly lowered the response rate of the electrochemotherapy treated tumours. The acquired intrinsic resistance of tumour cells to chemotherapeutics could be one of the possible reasons for the observed, as recurrent tumours emerge from those resistant tumor cells that survived previous radiation therapy due to certain adaptations, like higher level of DNA damage repair. It is less known, how those adaptations affect the chemoresistance of tumour cells, and consequently the resistance of those cells to electrochemotherapy. Aim: In the dissertation, our aim was to explore mechanisms of intrinsic chemo- and radioresistance of reccurrent tumours after previous irradiation, which could affect the effectiveness of electrochemotherapy. Hypothesis: Two hypotheses were explored. (1) Radioresistant tumour cells are less responsive to electrochemotherapy due to intrinsic resistance to cisplatin or bleomycin and (2) The development of chemoresistance in radioresistant tumour cells is a result of difference in DNA repair mechanisms and altered cellular uptake of chemotherapeutics. Materials and methods: In our experiments, we compared the two human cell lines of squamous cell carcinoma of the pharynx: the parental (FaDu) and the radioresistant subline (FaDu-RR) that was established by fractionated irradiation of the FaDu line. With the clonogenic test, we first determined the degree of sensitivity of both cell lines to irradiation, continuous exposure to cisplatin or bleomycin, and to electrochemotherapy. To perform in vivo experiments, we induced FaDu and FaDu-RR tumours by subcutaneous injection of the two cell lines on the right flanks of the immunocompromised (SCID) mice. We compared the tumour models in some histological characteristics and in response to irradiation, concomitant irradiation with cisplatin, chemotherapy and to electrochemotherapy. The effect of the therapies was monitored by measuring the tumour growth delay, compared to control groups, and with survival analysis. In the second part of the study, we compared the two cell lines in capacity to repair DNA double strand breaks (by immunocytochemical determination of γH2AX foci) and in DNA repair signaling pathway gene expression (by PCR). Additionally, using highly specific mass spectrometry analytical methods, we determined the cisplatin or bleomycin tumour uptake after chemo- and electrochemotherapy. Results: With fractionated irradiation, we successfully established radioresistant subline FaDuRR that developed defense mechanisms leading to radio- and cisplatin cross-resistance. In vivo, we showed that FaDu-RR tumours were more resistant to concurrent radiochemotherapy with cisplatin, as well as to electrochemotherapy with cisplatin. Importantly, both cell lines and tumour models were equally sensitive to electrochemotherapy with bleomycin. In the second part of the study, where we investigated potential resistance mechanisms, we have demonstrated that in addition to the reduced intracellular accumulation of cisplatin compared to the parent cell line, the radioresistant cells also demonstrated a more efficient repair of DNA damage and have altered spectrum of DNA damage repair gene expression. Conclusions: Fractionated irradiation in the newly established cell line induced some adaptation mechanisms such as more effective repair of DNA damage and cross-resistance to cisplatin. In in vivo conditions, FaDu-RR tumours were equally sensitive to bleomycin-based electrochemotherapy and more resistant to cisplatin-based electrochemotherapy compared to FaDu tumours. Given the fact that the difference between tumour models was small, based on these results we can exclude a more significant role of intrinsic radioresistance of tumour cells in the response of previously irradiated tumours to electrochemotherapy. However, regarding a certain degree of cross-resistance in the radioresistant cells to cisplatin, the bleomycin-based electrochemotherapy should be considered in the treatment of previously irradiated tumours.

Izhodišče in namen: Incidenca primarnih jetrnih tumorjev v svetu se povečuje. Najpogostejši med njimi (90 %) je hepatocelični karcinom (HCC) in skupaj z intrahepatičnim holangiokarcinom (CHC) predstavlja 98,5 % primarnih jetrnih tumorjev. Ne glede na dejstvo, da je to eden najpogostejših vzrokov smrti zaradi raka v svetu, je manj kot 20 % bolnikov s HCC kandidatov za kurativno zdravljenje – kirurško resekcijo ali presaditev jeter. Samo kurativno zdravljenje omogoča bolnikom možnost za dolgoletno preživetje s petletnim preživetjem do 70 % pri kirurških resekcijah in presaditvi jeter. Ko radikalni/kurativni tretmaji zaradi splošnega stanja telesne zmogljivosti, pridružene okvare jeter ali velikosti in razširjenosti tumorja ne pridejo v poštev, uporabljamo druge metode, kot sta transarterijska kemoembolizacija (TACE) in transarterijska radioembolizacija (TARE), ali lokalne ablativne tehnike, kot so radiofrekvenčna ablacija (RFA), mikrovalovna ablacija (MWA), krioterapija in druge metode za zdravljenje primarnih jetrnih tumorjev, ki podaljšajo preživetje teh bolnikov. Elektrokemoterapija (ECT) omogoča netoplotno ablacijo kožnih in tudi globlje ležečih tumorjev. Na podlagi dobrih rezultatov pri zdravljenju jetrnih zasevkov raka debelega črevesa in danke smo opravili prospektivno raziskavo zdravljenja hepatoceličnega karcinoma z namenom ugotavljanja izvedljivosti, varnosti in učinkovitosti elektrokemoterapije pri primarnih jetrnih tumorjih. Metode: Elektrokemoterapijo z bleomicinom smo izvedli pri 25 bolnikih s hepatoceličnim karcinomom, skupno je bilo tretiranih 33 lezij po predvidenem protokolu. Zdravljenje je bilo izvedeno med odprto laparotomijo pri 24 od 25 bolnikov, pri enem bolniku je bila elektrokemoterapija izvedena perkutano pod kontrolo računalniške tomografije. Ob zaključku spremljanja bolnikov je bila mediana opazovanja 23,5 meseca. Rezultati: Elektrokemoterapija je bila izvedljiva pri vseh 33 hepatoceličnih karcinomih. Pri nobenem bolniku ni prišlo do neželenih stranskih učinkov zdravljenja ali večjih pooperativnih zapletov. Do popolnega odgovora na zdravljenje je po 3 do 6 mesecih prišlo pri 92 % bolnikov in 94 % zdravljenih lezij. Ob zaključku spremljanja, tj. po medianem času spremljanja 23,5 meseca, je pri 31 od 33 lezij prišlo do popolnega odgovora. Zaključki: Elektrokemoterapija primarnih jetrnih tumorjev se je izkazala kot izvedljiv in varen način zdravljenja pri vseh 25 bolnikih. Za dokončno oceno učinkovitosti zdravljenja je potreben daljši interval sledenja bolnikov. Elektrokemoterapija je predvsem primerna pri bolnikih z zmanjšano jetrno funkcijo zaradi jetrne ciroze, pri bolnikih z zmerno do hudo portalno hipertenzijo in/ali pri zdravljenju lezij, pri katerih bi lahko zgolj operacija z velikim tveganjem za zaplete ponudila možnost popolne ozdravitve, saj elektrokemoterapija ponuja možnost popolnega odgovora lezije na zdravljenje. Background and objectives: The incidence of primary liver cancer (PLC) is rising across the world. Hepatocellular carcinoma (HCC) is the most common (90%) and it and intrahepatic cholangiocarcinoma represent more than 98.5% of all primary liver tumors. Despite being one of the most common causes of cancer-related death around the world, fewer than 20%of patients with HCC are eligible for curative treatment—that is, surgical resection or liver transplantation. Only curative treatment offers the patient a chance for long-term survival, with 5-year survival rates of 70% for liver resection and liver transplantation. When radical or curative treatment is not indicated due to the patient’s condition, underlying liver disease, or tumor extent, other methods such as transarterial chemoembolization (TACE), transarterial radioembolization (TARE), and local ablative treatments such as radiofrequency ablation (RFA), microwave ablation (MWA), cryotherapy, and other methods help in longer survival of those patients. Electrochemotherapy provides non-thermal ablation of cutaneous and deep-seated tumors. Based on positive results of the treatment of colorectal liver metastases, we conducted a prospective pilot study on hepatocellular carcinomas with the aim of testing the feasibility, safety, and effectiveness of electrochemotherapy. Patients and methods: Electrochemotherapy with bleomycin was performed on 33 hepatocellular carcinomas in 25 patients using a previously established protocol. The procedure was performed during open laparotomy for 24/25 patients, and percutaneous electrochemotherapy with computed tomography control was performed for one patient. At the end of the observation period, the median follow-up was 23.5 months. Results: Electrochemotherapy was feasible for all 33 lesions, and no treatment-related adverse events or major postoperative complications were observed. The complete response rate at 3 to 6 months was 92% per patient and 94% per treated lesion. At the end of the observation period, after a median follow-up time of 23.5 months, complete response was obtained in 31 out of 33 lesions. Conclusions: Electrochemotherapy of hepatocellular carcinoma proved to be a feasible and safe treatment in all 25 patients. To evaluate the effectiveness of this method, a longer observation period is needed. Electrochemotherapy is predominantly applicable for patients with impaired liver function due to liver cirrhosis, in patients with mild to severe portal hypertension, and/or for lesions where a high-risk operation is needed to achieve curative intent because ECT offers the possibility for complete response of the lesion to treatment.

Elektrokemoterapija je kombinirana fizikalno-kemijska metoda, pri kateri z izpostavitvijo celic ali tkiva električnim pulzom povzročimo privzem citostatikov v celico. Je lokalna metoda zdravljenja z malo neželenimi učinki in nizko toksičnostjo. Kot najprimernejši zdravili sta se do sedaj izkazala bleomicin in cisplatin. Bleomicin je protitumorsko zdravilo, ki prek tvorbe dvojnih prelomov ustavlja celične delitve in povzroča smrt celic, cisplatin pa je alkilirajoči agens, ki v celicah primarno tvori adukte DNA, s tem pa prepreči podvojevanje DNA in nadaljnje delitve celic. V magistrski nalogi nas je zanimalo, ali se citotoksični učinek elektrokemoterapije poveča, če elektrokemoterapijo kombiniramo z olaparibom. Olaparib namreč zavirala popravljalni mehanizem PARP, ki ob nastanku enojnih in dvojnih prelomov rekrutira popravljalne mehanizme in omogoči popravljanje poškodb DNA. Citotoksično delovanje kombinacije smo preverili na humani celični liniji MCF-7 raka dojke, na 2D in 3D celičnih kulturah. Obetavne rezultate je prinesla kombinacija olapariba z elektrokemoterapijo z bleomicinom. Z barvanjem po Giemsi smo določili nastale morfološke spremembe. Z detekcijo žarišč ?-H2AX pa smo potrdili nastanek dvojnih prelomov po elektrokemoterapiji celic z bleomicinom in kombinaciji elektrokemoterapije z bleomicinom in olaparibom. Electrochemotherapy is a physical delivery method, where cells or tissues are exposed to electric pulses, which increase the uptake of cytotoxic drugs into cells. It is a local ablative treatment method with almost negligible side effects and high antitumor effectiveness. Among many tested substances, only bleomycin and cisplatin have been mainly used. Bleomycin is an anti-cancer drug, which inhibits cell divisions by forming double strand breaks, whereas cisplatin is an alkylating agent that forms DNA aducts and inhibits DNA replication and cell divisions. The aim of the study was to investigate if the combination of electrochemotherapy with bleomycin or cisplatin and olaparib, which is a PARP inhibitor, has a greater cytotoxic effect than only electrochemotherapy. PARP is a repair mechanism that recruits other repair mechanisms when single and double strand breaks occur. We evaluated the effects of the combined treatment on human breast cancer cell line MCF-7. We observed promising effects of combining electrochemotherapy with bleomycin and olaparib, proving that the combination highly decreases cancer cell growth. We used 2D and 3D cell cultures. Giemsa staining showed morphological changes, whereas the detection of ?-H2AX confirmed formation of double strand breaks after electrochemotherapy with bleomycin alone or in combination with olaparib.

Rak trebušne slinavke velja za enega najbolj smrtonosnih rakov. Srednje preživetje je zaradi poznega odkritja le 3 – 6 mesecev. V primeru zgodnjega odkritja raka, se tumor operativno odstrani, sicer pa se bolnikom podaljšuje življenje s kemoterapijo, pri čemer se običajno uporabi gemcitabin. Lahko se zdravi tudi z radioterapijo, kjer z obsevanjem poskušajo izzvati mutacije v celici, ki privedejo do apoptoze. Zdravljenje raka trebušne slinavke je zahtevno zaradi gostega tumorskega mikrookolja, poleg tega pa zaviralci imunskih kontrolnih točk zavirajo citotoksično delovanje limfocitov T. Novi načini zdravljenja se zato osredotočajo predvsem na imunoterapijo. Zaviralci kontrolnih točk, kot so PD-L1, CTLA-4 in VISTA, so humana transgena monoklonska protitelesa, ki jih običajno proizvajajo z transgenimi CHO celicami. Izsledki raziskav kažejo, da terapija samo z zaviralci imunskih kontrolnih točk ni učinkovita, so pa veliko uspešnejše kombinirane terapije, predvsem v kombinaciji s cepivi ali kemoterapijo. Največji uspeh je dosegla terapija s cepivi z dendritičnimi celicami, ki jih izpostavijo tumorskim antigenom, v kombinaciji s kemoterapijo, ob uporabi te terapije se preživetje podaljša na 16,7 mesecev, kombinacija cepiv z zaviralci imunskih kontrolnih točk pa le na 6 mesecev. Terapija s CAR T celicami še ni dobro razvita, so pa raziskave z elektrokemoterapijo pokazale, da imajo celice raka trebušne slinavke visoko izražene transkripcijske markerje značilne za matične celice. Pancreatic cancer is said to be one of the deadliest types of cancer with median survival of 3 – 6 months due to late diagnosis. When the cancer is found early, it can be operable, however patients are usually diagnosed with stage III or IV pancreatic cancer. In such cases, chemotherapy is used to prolong survival, gemcitabine being the most commonly used chemotherapeutic, followed by bleomycin and cisplatin. Rarely it is treated with radiotherapy, where radiation is used to induce mutations that lead to apoptosis. Treatment of pancreatic adenocarcinoma is difficult due to thick tumor microenvironment. Immune checkpoint inhibitors also inhibit the cytotoxic properties of lymphocytes T. Therefore, new treatment methods are focused on immunotherapy. Human monoclonal antibodies produced in CHO cell lines, that inhibit immune checkpoints, such as PD-L1, CTLA-4 and VISTA, are not showing much success by themselves, but different combinatorial therapies are more promising. Specifically, vaccines with dendritic cells exposed to tumor antigens prolong combined with chemotherapy prolong life for a full year, while the combination with immune checkpoint inhibitors only prolongs survival for 6 months. CAR T therapy is not well developed yet, however electrochemotherapy is bringing life to new discoveries, proving high level of stemness markers in pancreatic cancer cells.

The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, non-randomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the follow-up protocol (7, 14 and 28 days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6 months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22 months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (Treg) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost.

Znanstveno izhodišče: Za razliko od začetnih stadijev raka, zaradi agresivnosti in odpornosti na terapije uspešnost zdravljenja malignega melanoma še vedno ne dosega pričakovanj. Novi terapevtski pristopi temeljijo predvsem na kombiniranju že uveljavljenih terapij z novejšimi terapijami, ki vplivajo na komponente imunskega sistema. Medtem ko je elektrokemoterapija s cisplatinom že dobro uveljavljena v kliniki, elektrokemoterapija z oksaliplatinom do danes še ni bila raziskana. Oksaliplatin je primeren kandidat za vpeljavo v elektrokemoterapijo zaradi podobnosti s cisplatinom, opisanih manjših stranskih učinkov in predpostavljeno večjega imunomodulatornega vpliva. Čeprav v kliniki z elektrokemoterapijo pri 80 % tumorjev dosežemo lokalno tumorsko kontrolo, protitumorski učinek na oddaljene nezdravljene tumorje in/ali metastaze (t. i. »abscopal effect«) še ni bil opisan. Namen doktorske naloge je bil preučiti učinkovitost intratumoralne elektrokemoterapije z oksaliplatinom in jo primerjati z elektrokemoterapijo s cisplatinom na mišjem malignem melanomu B16F10. Preverili smo tudi, v kolikšni meri lahko peritumoralni genski elektroprenos plazmida z zapisom za interlevkin-12 (IL-12) poveča lokalno učinkovitost elektrokemoterapije in ali lahko spodbudi tudi sistemski odgovor, to je protitumorski učinek na oddaljene nezdravljene tumorje. Metode: Raziskave so potekale na celični liniji mišjega malignega melanoma B16F10. V prvem, in vitro delu smo raziskali, ali električni pulzi potencirajo privzem citostatika oksaliplatina v celice ter vezavo platine na DNA. Vsebnost platine v vzorcih smo izmerili s pomočjo masne spektrometrije z induktivno sklopljeno plazmo. Zanimala nas je tudi potenciacija v citotoksičnosti ob aplikaciji električnih pulzov in primerjava občutljivosti celic B16F10 na oksaliplatin in cisplatin. Dokazali smo jo s testom klonogenosti. Citometrično smo določili vrsto celične smrti oziroma delež celic, ki umirajo z apoptozo, nekrozo ali imunogeno celično smrtjo. Nadaljevali smo s poskusi na miših C57Bl/6. Tumorske celice mišjega melanoma B16F10 so po injiciranju v podkožje na boku miši tvorile čvrste tumorje. S terapijo smo začeli, ko so tumorji dosegli 35&#61485 40 mm3. Preverili smo protitumorsko učinkovitost elektrokemoterapije z oksaliplatinom in jo primerjali z elektrokemoterapijo s cisplatinom. Kombinirano terapijo sta sestavljala peritumoralni genski elektroprenos plazmida z zapisom za IL-12 in intratumoralna elektrokemoterapija s cisplatinom ali oksaliplatinom. Najprej smo izvedli genski elektroprenos, in sicer tako, da smo v štiri mesta peritumoralno intradermalno injicirali plazmid (skupaj 50 µg). Nizkonapetostne električne pulze (12 pulzov, 170 V/cm, 150 ms, 0,4/1,39 Hz) smo dovedli z več-točkovno elektrodo (MEA) s krožno razporeditvijo šestih elektrodnih konic takoj po injiciranju plazmida. Pet minut po genski terapiji smo intratumoralno injicirali cisplatin ali oksaliplatin, po dveh minutah pa dovedli električne pulze s ploščatimi elektrodami (8 pulzov, 1300 V/cm, 100 µs, 1 Hz). Preverili smo, ali dodatne ponovitve genskega elektroprenosa IL-12 izboljšajo protitumorski učinek kombinirane terapije. Za določanje sistemskega vpliva kombinirane terapije oziroma vpliva na oddaljene nezdravljene tumorje smo uvedli model za sistemsko bolezen. Tumorske celice smo injicirali v oba boka živali v desni bok 1 x 106 celic, v levi bok pa 0,7 x 106 celic. Med izvedbo elektrokemoterapije in genskega elektroprenosa so bile miši pod inhalacijsko anestezijo z izofluranom. Zdravili smo desni tumor, zaostanek v rasti pa spremljali na obeh tumorjih, zdravljenem in nezdravljenem. Protitumorski učinek oziroma zaostanek v rasti tumorjev smo spremljali s kljunastim merilom. Za določanje koncentracije IL-12 v serumu, vsebnosti platine v tumorjih in serumu ter histološko analizo smo po humani usmrtitvi živali odvzeli tumorje in kri. Imunohistokemijsko smo označili nekrozo in apoptozo (kaspaza 3), izražanje transgena (IL-12) in populacije imunskih celic v tumorju (grancim B, Foxp3). Rezultati: Citotoksičnost oksaliplatina v kombinaciji z električnimi pulzi je bila visoka in primerljiva s citotoksičnostjo cisplatina na celicah mišjega melanoma B16F10 in vitro. Primerljiva citotoksičnost je bila posledica primerljive potenciacije v privzemu platine v celice in vezavi platine na DNA. Na melanomskem modelu in vivo smo pokazali, da je elektrokemoterapija z oksaliplatinom enako učinkovita kot elektrokemoterapija s cisplatinom, če dozo oksaliplatina dvignemo za faktor 1,6. Izpostavitev melanomskih celic elektrokemoterapiji z oksaliplatinom ali cisplatinom je vodila tudi v imunogeno celično smrt, slednje pa je koreliralo s povišano infiltracijo imunskih celic v tumor po terapiji. Po elektrokemoterapiji z nizkimi dozami oksaliplatina ali cisplatina smo opazili le zaostanek v rasti primarnih tumorjev, ne pa tudi popolnih odgovorov. Pokazali smo, da peritumoralni genski elektroprenos plazmida z zapisom za IL-12 potencira protitumorski učinek intratumoralne elektrokemoterapije s cisplatinom ali oksaliplatinom na mišjem malignem melanomu B16F10. Ob enako učinkoviti elektrokemoterapiji s cisplatinom ali oksaliplatinom je bil prispevek genskega elektroprenosa IL-12 večji pri elektrokemoterapiji s cisplatinom, dosegli smo namreč 38 % popolnih odgovorov. Po kombinirani terapiji z oksaliplatinom smo opazili le značilen zaostanek v rasti tumorjev. Dodatno ponavljanje genskega elektroprenosa plazmida z zapisom za IL-12 ni izboljšalo terapevtskega učinka, povzročilo je tudi infiltracijo peritumoralnega prostora s celicami T zaviralkami. Najpomembneje, kombinirana terapija, pri kateri smo za elektrokemoterapijo uporabili cisplatin, je imela poleg lokalnega tudi sistemski učinek, dvodnevni zaostanek v rasti oddaljenih nezdravljenih tumorjev. Za uspešnost opisane kombinirane terapije je najverjetneje odgovorno lokalno in sistemsko spodbujanje imunskega odziva. Po kombinirani terapiji s cisplatinom v elektrokemoterapiji smo namreč v zdravljenih in nezdravljenih tumorjih opazili imunske celice, pozitivne za grancim B. Zaključki: Genski elektroprenos plazmida z zapisom za IL-12 je učinkovit pri spodbujanju lokalnega protitumorskega učinka elektrokemoterapije. Poleg tega ima kombinirana terapija, pri kateri smo za elektrokemoterapijo uporabili cisplatin, tudi sistemski protitumorski učinek. Rezultati doktorske naloge bodo v pomoč pri vpeljevanju oksaliplatina v elektrokemoterapijo, kot samostojno terapijo, v kliniko. Rezultati v sklopu testiranja kombinirane terapije pa bodo osnova za prehod na naslednjo stopnjo v verigi translacijskih raziskav, prijavo klinične študije. Scientific background: Treatment of malignant melanoma remains a challenge due to its aggressiveness and resistance to treatment. Combining established local ablative therapies with recently discovered immunotherapies is a promising approach for treating malignant melanoma. While electrochemotherapy with cisplatin is already a well-established treatment, oxaliplatin has not yet been tested for use with electrochemotherapy. Based on its similar characteristics to cisplatin, described lesser side-effect and its presumably more pronounced immunomodulatory effect, oxaliplatin is an eligible candidate for electrochemotherapy. Although local effectiveness of electrochemotherapy is up to 80 % of local tumor control, noticeable effects on distant non-treated metastases (abscopal effect) have yet to be described. With the aim to increase the armamentarium of drugs for electrochemotherapy, oxaliplatin was compared to the already well-established electrochemotherapy with cisplatin in murine malignant melanoma B16F10. In addition, we also investigated if peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (IL-12) as an adjuvant immunotherapy enhances the responses seen with electrochemotherapy on a local and systemic level (abscopal effect). Methods: Murine B16F10 melanoma cells with a high metastatic potential were used. Firstly, we determined the potentiation in the uptake of platinum after applying electric pulses and platinum-based drugs oxaliplatin or cisplatin in vitro. Platinum content in cells and the amount of platinum bound to DNA were measured by inductively coupled plasma mass spectrometry. Using the clonogenic assay we determined the cellular reproductive potential following electroporation with oxaliplatin or cisplatin. We also determined the type of cell death after electroporation with oxaliplatin or cisplatin. More specifically, we determined the amount of cells which died via apoptosis, necrosis or immunogenic cell death. Secondly, in vivo experiments were performed in C57Bl/6 mice. Solid tumors were grown on the right flank of mice after a subcutaneous inoculation of tumor cells. The therapy was performed when tumors reached 35&#61485 40 mm3 in volume. The antitumor effect of electrochemotherapy was compared to electrochemotherapy with cisplatin. Thereafter, the antitumor effect of the combined treatment consisting of intratumoral electrochemotherapy with cisplatin or oxaliplatin and peritumoral gene electrotransfer of plasmid encoding IL-12 was researched. For gene electrotransfer mice received four intradermal injections of plasmid DNA encoding IL-12 (50 µg) around the tumor. Immediately after low voltage electric pulses (12 pulses, 170 V/cm, 150 ms, 0.4/1.39 Hz) were applied with a multi-electrode array (MEA). Five minutes after gene electrotransfer, oxaliplatin or cisplatin were injected intratumorally and after two minutes electric pulses (8 pulses, 1300 V/cm, 100 µs, 1 Hz) were applied with plate electrodes. The antitumor effectiveness of the combined treatment with multiple repetitions of gene electrotransfer was also tested. A dual flank model mimicking systemic disease was used to determine the systemic antitumor effect of the therapy. It was established by subcutaneous injections of tumor cells on both flanks of mice, 1 x 106 cells on the right and 0.7 x 106 on the left flank. During the treatment, animals were under inhalational anesthesia with isoflurane. Tumor growth was followed by measuring three mutually orthogonal tumor diameters with a Vernier caliper. The right tumor was regarded as a primary tumor and was treated the left tumor was left untreated. Blood samples were collected for the detection of IL-12 in mice sera and tumor samples were collected for histological analysis. Immunohistochemical analysis was performed to detect necrosis, apoptosis (caspase 3), transgene expression (IL-12) and infiltration of immune cells into tumors (granzyme B, Foxp3). Results: Electroporation potentiated the cytotoxicity of oxaliplatin in vitro on B16F10 melanoma cells and no differences in cytotoxicity between cisplatin and oxaliplatin were observed. Cytotoxicity correlated with cisplatin or oxaliplatin uptake into cells and drug binding to DNA. Based on in vivo results pertaining to effectiveness and drug uptake in tumors, electrochemotherapy with oxaliplatin is as effective as electrochemotherapy with cisplatin when the dose of oxaliplatin is increased 1.6-fold. Electrochemotherapy induced immunogenic cell death when either oxaliplatin or cisplatin were used, which correlated with a comparable increase in lymphocyte infiltration into tumors after electrochemotherapy with either oxaliplatin or cisplatin. We observed a delay in tumor growth after electrochemotherapy with low doses of oxaliplatin or cisplatin, with no complete responses. We showed that peritumoral gene electrotransfer of plasmid encoding IL-12 potentiated the antitumor effect of electrochemotherapy with oxaliplatin or cisplatin in B16F10 melanoma on a local level. The most pronounced potentiation was with electrochemotherapy using cisplatin, resulting in 38% of complete responses. However, the additional repetitions of gene electrotransfer did not improve the therapeutic effectiveness. Moreover, we also observed an abscopal effect after the combination treatment with electrochemotherapy with cisplatin. Average tumor growth delay of the distant untreated tumors in the latter group was two days. We ascribe the success of this novel therapy to the induction of an immune response on a local and systemic level. Namely, infiltration of granzyme B positive immune cells after the combination therapy with electrochemotherapy with cisplatin was observed in both primary and distant tumors. Conclusion: We showed that gene electrotransfer of IL-12 potentiates the effect of electrochemotherapy on a local level. Additionally, an abscopal effect was detected after the combination therapy in which cisplatin was used for electrochemotherapy. Our findings highlight the potential clinical use of intratumoral oxaliplatin in electrochemotherapy. Most importantly, the preclinical data will serve as the basis for a clinical phase study application.

Znanstveno izhodišče: Elektrokemoterapija je inovativen način zdravljenja raka, vendar je malo raziskav, ki bi bile osredotočene na njeno vlogo v zdravljenju sluzničnega in kožnega raka glave in vratu. Za ugotavljanje učinkovitosti in varnosti zdravljenja tudi pri tej skupini bolnikov so nujne pravilno zasnovane in izvedene prospektivne klinične raziskave. Z izboljšanjem tehnoloških postopkov zdravljenja z elektrokemoterapijo lahko zdravimo tudi globoko ležeče tumorje glave in vratu. Pri zdravljenju z elektrokemoterapijo se najpogosteje uporablja bleomicin. Vendar lahko sistemsko zdravljenje z bleomicinom izzove stranske učinke, predvsem pljučno fibrozo in razjede na mestih zdravljenja. Da bi bilo zdravljenje z elektrokemoterapijo kar najbolj učinkovito in varno, je poleg ostalega potrebno določiti tudi parametre farmakokinetike bleomicina. Prav tako je potrebno razjasniti vpliv tumorskega mikrookolja na porazdelitev bleomicina v tumorju in posledično na učinkovitost zdravljenja z elektrokemoterapijo. Namen dela: Namen doktorskega dela je bil ovrednotiti učinkovitost in varnost elektrokemoterapije z bleomicinom v zdravljenju raka glave in vratu. Specifični cilji so bili: (i) določitev optimalnega časovnega okvirja od časa injiciranja bleomicina do elektroporacije in optimalne koncentracije bleomicina, ki je potrebna za učinkovito elektrokemoterapijo, (ii) izboljšanje tehnološkega postopka zdravljenja globoko ležečih tumorjev v področju glave in vratu, (iii) določitev napovednih dejavnikov za uspešnost elektrokemoterapije. Hipoteza: Elektrokemoterapija je učinkovita in varna metoda zdravljenja raka v področju glave in vratu. Metode: V doktorski disertaciji smo spremljali učinek in varnost elektrokemoterapije pri bolnikih s kožnim in sluzničnim rakom glave in vratu. S sklopitvijo načrta zdravljenja in navigacijskega sistema smo poskušali izboljšati postopek zdravljenja globoko ležečih tumorjev v področju glave in vratu. Parametre farmakokinetike bleomicina smo določili tako pri bolnikih kot in vivo pri miših s tekočinsko kromatografijo sklopljeno z masno spektrometrijo. Tumorsko mikrookolje smo preučevali z imunohistokemično analizo na karcinomskem in melanomskem tumorskem modelu. Rezultati: S spremljanjem odgovora na zdravljenje smo dokazali, da je elektrokemoterapija varno in učinkovito zdravljenje raka glave in vratu. Pokazali smo, da je z optimizacijo tehnoloških postopkov elektrokemoterapije možno učinkovito in varno zdraviti tudi v globini ležeče tumorje glave in vratu. Pri starejših bolnikih je terapevtsko okno za aplikacijo električnih pulzov po intravenoznem injiciranju bleomicina daljše kot pri mlajših. Hkrati je pri njih nižji odmerek bleomicina enako učinkovit kot standardni odmerek. Odgovor na zdravljenje z elekokemoterapijo je odvisen od ožiljenosti tumorja, ki vpliva na razporeditev bleomicina v tumorju. Zaključki: Elektrokemoterapija se je izkazala za učinkovit in varen način zdravljenje raka v različnih predelih glave in vratu. Rezultati raziskave pomembno prispevajo k širjenju terapevtskih možnosti pri tem raku in k prilagoditvi elektrokemoterapevtskih protokolov posameznemu bolniku. Značilnosti tumorskega mikrookolja so pomemben dejavnik za napoved uspešnosti elektrokemoterapije. Scientific background: Electrochemotherapy is an inventive approach to cancer treatment. Howevwer, only a few studies so far have focused on its role in the treatment of skin and mucosal head and neck cancers. Properly designed and well-conducted prospective clinical trials are required to determine the efficiency and safety of electrochemotherapy in this group of patients. Technological improvements implement electrochemotherapy as a feasible treatment also for deep-seated head and neck tumors. Nevertheless, intravenously administrated bleomycin is currently the most utilized drug for electrochemotherapy, but it might have some specific side effects, predominantly lung fibrosis and possible ulceration in the treated areas. The main parameters of bleomycin pharmacokinetics need to be determined in order to provide optimal effectiveness and safety of electrochemotherapy. Furthermore, the role of tumor microenvironment in bleomycin distribution and its consequential impact on tumor response to electrochemotherapy has to be explained. Aim: The aim of the study was to evaluate the effectiveness and safety of bleomycin-based electrochemotherapy in the treatment of head and neck cancer. Specific objectives were: (i) to determine an optimal time frame between bleomycin injection and electroporation and optimal bleomycin concentration required for effective electrochemotherapy, (ii) the improvement of technological procedure in the treatment of deep-seated head and neck tumors and (iii) determination of predictive factors for effective electrochemotherapy. Hypothesis: Electrochemotherapy is an effective and safe treatment modality for the treatment of head and neck cancer. Methods: In the scope of the study, the local anti-cancer effectiveness and safety were followed on the skin and mucosal head and neck cancers treated with electrochemotherapy. The efficiency of electrochemotherapy as a treatment option of deep-seated head and neck tumors was evaluated by coupling treatment planning and navigation system. The parameters of bleomycin pharmacokinetics were determined in patients and in vivo in mice by liquid chromatography coupled with high-resolution mass spectrometry. Tumor microenvironment on carcinoma and melanoma tumor models was additionaly investigated with immunohistochemical analysis. Results: The evaluation of treatment outcome in patients with head and neck cancer treated with electrochemotherapy confirms the efficacy and safety of electrochemotherapy in this group of patients. The optimized technological approach enables the treatment of deep-seated tumors in the head and neck region. In the group of elderly patients, therapeutic window is prolonged compared to younger patients and a reduced dose of bleomycin is equally effective as a standard dose. The tumor response to electrochemotherapy is influenced by tumor vascularization, which affects bleomycin distribution in the tumor. Conclusion: Electrochemotherapy proved to be an efficient and safe treatment of cancers in different head and neck regions. The results of the study broaden therapeutic options in the treatment of head and neck cancer and contribute to the adaptation of the electrochemotherapeutic protocols to each individual patient. Characteristics of tumor microenvironment are an important predictive factor to the tumor response to electrochemotherapy.

Purpose Electrochemotherapy is increasingly entering into national and international guidelines, requiring formal evaluation of treatment costs and cost-effectiveness to ensure that its uptake provides value to budget-constrained health care systems. This study analyzed the early cost-effectiveness of electrochemotherapy in patients with Stage IIIc/IV skin melanoma in clinical practice in Slovenia. The costs of electrochemotherapy were compared to those of the standard of care, consisting of palliative treatment and therapy for symptoms. Methods wThe study enrolled 23 patients treated with electrochemotherapy at the Institute of Oncology (Ljubljana, Slovenia). The mean cost of electrochemotherapy was estimated using patient-specific cost data on electrochemotherapy procedures and subsequent follow-up. Quality-adjusted life-years (QALYs) were estimated by collecting EQ-5D-3L questionnaires at baseline, after complete or partial response following the treatment, and after a relapse of skin lesions. A discrete-time Markov model was built to estimate the lifetime costs and consequences of using electrochemotherapy compared to standard of care, from the perspective of the Slovenian health care system. The analysis was conducted separately in the whole patient sample and in the subset of patients with bleeding lesions. Deterministic and probabilistic sensitivity analyses were conducted to test model assumptions and to characterize the uncertainty around model parameters. Findings In the whole patient population, electrochemotherapy for skin melanoma Stage IIIc/IV was expected to increase QALYs by 0.29 (95% credible interval [CrI], 0.10–0.50), at the higher cost of 6568 EUR (95% CrI, 4593–8928) in comparison to the standard of care. At the cost-effectiveness threshold of 20,000 EUR/QALY, the estimated probabilities of electrochemotherapy being cost-effective compared to standard of care were 0.30 and 0.91 in the whole patient sample and in patients with bleeding lesions, respectively. In the whole sample population, a 50% reduction in the price of the electrodes was expected to increase the probability of electrochemotherapy being cost-effective from 0.30 to ~0.64. Implications The findings from this cost-effectiveness analysis of data from clinical practice were based on a small sample size (ie, 23 patents), which made the subgroup of patients with bleeding lesions very small. Therefore, the findings in this patient population should be carefully interpreted.

In the master's thesis, we investigated the possible effects of high-voltage electroporation pulses on the functioning of the pacemaker, under various conditions and methods of pulse delivery. We also developed simulation models that would allow us a more systematic study and understanding of physical phenomena and facilitate further research We were interested in how the influence of the pulses depends on the horizontal distance of the electrodes of the Cliniporator to the electrodes of the pacemaker, what are the maximum currents and voltages that can occur on the pacemaker electrodes and what are the effects when the Cliniporator pulses are delivered at different stages of pacing of the pacemaker. Measurements were carried out in a glass container, with a medium for two different values of electrical conductivity. The results obtained can be compared with voltages and currents that are caused in the body by other medical devices, for which it was proven that they cannot damage the pacemaker or cause its malfunction. While carrying out the measurements, we also came to a realization, that during the delivery of the Cliniporator pulses, the property of the media changed locally as evidenced by the sparking observed at the electrodes of the Cliniporator. Using the results of experimental measurements, two simulation models were constructed. The first model was constructed using linear electrical lumped elements, which were calculated by making the assumptions about the physical properties of the experimental setup during the measurements and using the results of these measurements. The second model was a numerical model using the finite element method, which required the proper composition of 3D geometry and the use of the output voltage of the Cliniporator. The Cliniporator pulse can affect the shape and amplitude of the pacemaker pulse, but the pacemaker pulse returns to its normal form in the next delivery cycle. In clinical circumstances, this should not present a problem, as the delivery of Clinipator pulses during electrochemotherapy are synchronized with the heart cycle and as such the Cliniporator pulses can never coincide with the pacemaker pulses. After all experimentation there was no change in the functioning of the pacemaker. The energy of the Cliniporator pulses delivered into the system is comparable to the energy delivered during defibrillation. Therefore, electrochemotherapy should be safe for patients with a pacemaker, taking similar precautions as for defibrillation. In order to confirm this, further examination of standards concerning cardiac pacemaker devices is needed. The simulation models achieved satisfactory results when computed signals were compared with the measured current and voltage signals. However, we should be aware of the inaccuracies introduced into the model with all the assumptions and simplifications used in building the models. V magistrski nalogi smo raziskovali možne vplive visoko napetostnih elektroporacijskih pulzov na delovanje srčnega spodbujevalnika, pod različnimi pogoji in načini dovajanja pulzov. Razvili smo tudi simulacijske modele, ki bi omogočili bolj sistematično preučevanje in razumevanje fizikalnih pojavov in olajšali nadaljnje raziskovanje. Zanimalo nas je, kakšen je vpliv pulzov v odvisnosti od horizontalne razdalje Cliniporatorjevih elektrod do elektrod srčnega spodbujevalnika, kakšni so maksimalni tokovi in napetosti, ki se lahko pojavijo na elektrodah spodbujevalnika, in kakšni so vplivi, kadar so Cliniporatorjevi pulzi dovedeni ob različnih fazah delovanja spodbujevalnika. Meritve smo izvajali v stekleni kadi z medijem pri dveh različnih vrednostih električne prevodnosti. Pridobljene rezultate lahko primerjamo z napetostmi in tokovi, ki jih v telesu povzročijo naprave, za katere je dokazano, da ne morejo drastično škodovati srčnemu spodbujevalniku. Pri izvajanju meritev smo prišli tudi do spoznanja, da se med dovajanjem Cliniporatorjevih pulzov, lokalno spreminja lastnost uporabljenega medija, zaradi pojava iskrenja ob Cliniporatorjevih elektrodah. S pomočjo rezultatov eksperimentalnih meritev smo sestavili dva simulacijska modela. Prvi model je sestavljen iz koncentriranih linearnih električnih elementov. Koncentrirane elemente smo poračunali s pomočjo predpostavk o fizikalnih lastnosti merilne postavitve in rezultatov meritev. Drugi model je bil numerični model z uporabo metode končnih elementov, ki je upošteval pravilno 3D geometrijo in izhodno napetost Cliniporatorja. Cliniporatorjevi pulzi lahko pri delujočem srčnem spodbujevalniku vplivajo na obliko in amplitudo dovedenega pulza spodbujevalnika, vendar se ta povrne nazaj na svojo normalno oblika že v naslednjemu ciklu delovanja. V klinični razmerah to ne bi smelo biti problematično, saj je dovajanje Cliniporatorjevih pulzov med elektrokemoterapijo natančno sinhronizirano z delovanjem srca in zato pri pravilni sinhronizaciji Cliniporatorjevi pulzi ne bi bili nikoli dovedeni med stimulacijskim pulzom spodbujevalnika. Po končanih eksperimentalnih meritvah nismo opazili nobenih sprememb v delovanju srčnega spodbujevalnika Dovedene energije Cliniporatorjevih pulzov v sistem so primerljive oziroma manjše kot so energije pri defibrilaciiji. Torej bi elektrokemoterapija s tega vidika morala varna za paciente z vgrajenim spodbujevalnikom ob upoštevanju podobnih previdnostnih ukrepov kot pri defibrilaciji. Za potrditev tega bo potrebno še podrobno preučiti standarde, ki se nanašajo na srčne spodbujevalnike. Simulacijski modeli, so dosegli zadovoljive rezultate, kadar smo izračunane signale primerjali s izmerjenimi signali tokov in napetosti. Vendar se je treba zavedati, kakšne napake smo vnesli v model z vsemi predpostavkami in poenostavitvami.

Elektrokemoterapija je način zdravljenja raka, ki združuje uporabo standardnih kemoterapevtikov in dovedenih električnih pulzov na območje tumorja. Z električnimi pulzi povzročimo destabilizacijo celične membrane,in posledično omogočimo lažje vstopanje citostatikov v celico, ki imajo sicer slabo prehajanje skozi membrano. Tako se večkrat poveča citotoksičnost citostatikov, kot sta cisplatin ali bleomicin, s tem pa se poveča tudi njihova protitumorska učinkovitost, posebno na mestu kamor dovedemo električne pulze. Zaradi selektivno povečanega vnosa samo na območju tumorja je terapevtski indeks elektrokemoterapije zelo dober, dobra je namreč lokalna protitumorska učinkovitost brez stranskih pojavov, lokalnih ali sistemskih bodisi zaradi kemoterapevtikov ali električnih pulzov. Po številnih predkliničnih raziskavah je bila elektrokemoterapija preizkušena tudi v mnogih kliničnih raziskavah. Elektrokemoterapija je učinkovita na vrsti histološko različnih tumorskih nodulov v koži ali podkožju, s 60–80 % objektivnih odgovorov tumorjev. Sedaj je na voljo nov generator električnih pulzov s pripadajočimi elektrodami, pripravljeni pa so tudi standardni postopki za zdravljenje z elektrokemoterapijo. S tem je omogočeno, da elektrokemoterapija postane standardno zdravljenje v onkologiji za uspešen lokalni nadzor rasti kožnih in podkožnih lezij različnih vrst raka, in je plod skupnega dela več evropskih onkoloških centrov.