277 research outputs found
HaN-Seg: The head and neck organ-at-risk CT & MR segmentation dataset
No full textThe HaN-Seg: Head and Neck Organ-at-Risk CT & MR Segmentation Dataset is a publicly available dataset of anonymized head and neck (HaN) images of 42 patients that underwent both CT and T1-weighted MR imaging for the purpose of image-guided radiotherapy planning. In addition, the dataset also contains reference segmentations of 30 organs-at-risk (OARs) for CT images in the form of binary segmentation masks, which were obtained by curating manual pixel-wise expert image annotations. A full description of the HaN-Seg dataset can be found in:
G. Podobnik, P. Strojan, P. Peterlin, B. Ibragimov, T. Vrtovec, "HaN-Seg: The head and neck organ-at-risk CT & MR segmentation dataset", Medical Physics, 2023. https://doi.org/10.1002/mp.16197,
and any research originating from its usage is required to cite this paper.
In parallel with the release of the dataset, the HaN-Seg: The Head and Neck Organ-at-Risk CT & MR Segmentation Challenge is launched to promote the development of new and application of existing state-of-the-art fully automated techniques for OAR segmentation in the HaN region from CT images that exploit the information of multiple imaging modalities, in this case from CT and MR images. The task of the HaN-Seg challenge is to automatically segment up to 30 OARs in the HaN region from CT images in the devised test set, consisting of 14 CT and MR images of the same patients, given the availability of the training set (i.e. the herein publicly available HaN-Seg dataset), consisting of 42 CT and MR images of the same patients with reference 3D OAR binary segmentation masks for CT images.If you are using the HaN-Seg public training dataset, you are required to cite the following article:
G. Podobnik, P. Strojan, P. Peterlin, B. Ibragimov, T. Vrtovec, "HaN-Seg: The head and neck organ-at-risk CT & MR segmentation dataset", Medical Physics, 2023. https://doi.org/10.1002/mp.16197
@ARTICLE{HaNSeg_dataset,
author = {Ga\v{s}per Podobnik, Primo\v{z} Strojan, Primo\v{z} Peterlin, Bulat Ibragimov, Toma\{z} Vrtovec},
title = {{HaN-Seg}: {T}he head and neck organ-at-risk {CT} \& {MR} segmentation dataset},
journal = {Medical Physics},
year = {2023},
doi = {10.1002/mp.16197}
Assessment of set-up errors in radiotherapy of patients with head and neck cancer: the importance of individual headrest
No full textUvod: Sledenje interfrakcijskih premikov ima v radioterapiji glave in vratu še posebno velik pomen, ker lahko že majhni premiki povzročijo predoziranost kritičnih organov in poddoziranost tarče. Glavno vlogo pri zmanjševanju tako nastavitvenih napak, kot tudi varnostnih robov ima zanesljiva in ponovljiva imobilizacija, ki jo lahko zagotovimo tudi z uporabo individualnih podlag za glavo in vrat. Namen: Namen raziskave je ovrednotiti natančnost obsevanja bolnikov z rakom glave in vratu ob uporabi standardnih in individualnih podlag za pod glavo in vrat ter primerjati izmerjene premike portalnih slik s pomočjo analiziranih sistematičnih in naključnih napak ter izračunanega varnostnega roba po van Herkovi formuli. Metode dela: V raziskavo je bilo vključenih 120 bolnikov z rakom glave in vratu, obsevanih z radikalnim namenom in intenzitetno modulirano (IMRT) oz. volumetrično modulirano ločno terapijo (VMAT). Razporedili smo jih v 6 skupin glede na časovno obdobje obsevanja in obsevalni aparat. V skupinah 1 in 2 (leto 2014) so bili vključeni bolniki, pri katerih so bile uporabljene standardne podlage za glavo in vrat, v ostalih skupinah (3-6, leti 2015 in 2017) pa bolniki z individualnimi podlagami. S pomočjo interfrakcijskih premikov na elektronskih portalnih slikah (EPI) smo analizirali sistematično in naključno populacijsko napako ter velikost varnostnega roba od kliničnega (CTV) do planirnega tarčnega volumna (PTV). Rezultati: Rezultati kažejo odstopanje interfrakcijskih premikov v posteriorno in inferiorno smer. Največja razlika interfrakcijskih premikov med standardnimi in individualnimi podlagami se pojavlja v anteroposteriorni (AP) smeri, sledi ji superoinferiorna (SI), najmanjše razlike je mogoče opaziti v mediolateralni (ML) smeri. Z uporabo individualnih podlag se sistematične in naključne napake večinoma zmanjšujejo – od 0,1 do 0,5 mm. Zaradi manjših sistematičnih in naključnih napak se z uvedbo individualnih podlag posledično zmanjša tudi CTV-PTV varnostni rob in sicer znaša v AP smeri 3,3 mm, 2,6 mm v SI in 3,2 mm v ML smeri. S p-vrednostjo p<10-3 lahko potrdimo statistično značilne razlike med interfrakcijskimi premiki pri uporabi standardnih oz. individualnih podlag. Razprava in zaključek: Dokazali smo, da uporaba individualnih podlag vpliva na zmanjšanje povprečnih interfrakcijskih premikov in števila povprečnih premikov izocentra. Pri standardnih podlagah je bila izračunana večja sistematična in individualno napaka ter opaženo večje število nedopustnih napak. 4 mm varnostni rob zagotavlja, da 90% bolnikov prejme vsaj 95% minimalne kumulativne doze na CTV.Introduction: The importance of evaluating interfractional movements in head and neck radiotherapy is crucial, since even small displacement can produce overdose of critical structures nearby or underdose the target volume. A solution to reduce set-up errors and safety margins is repeatable and certain immobilization, such as usage of individual headrest. Purpose: The purpose of this research was to evaluate the importance of individual headrest in head and neck radiotherapy, to calculate interfractional movements, systematic and random error and to compare optimum safety margin between group with individual and standard headrest. Methods: In present study we included 120 patients with head and neck cancer, radically treated with intensity modulated (IMRT) or volumetric modulated radiation therapy (VMAT). They were divided into 6 groups, considering linear accelerator and time period in which they were treated. Groups 1 and 2 represented patient with standard headrest, and the rest of them, ones with individual. Systematic population and random population errors were evaluated, based on interfractional movements, that were measured on daily pre-treatment electronic portal images (EPI). Also the optimum safety margins of clinical and planning target volume, according to van Herk, were calculated. Results: The substantial interfraction movements show in posterior and inferior direction. The major difference between individual and standard headrest is represented in anteroposterior (AP) direction and inconsiderable in mediolateral (ML) direction. Since the individual headrest have been in use, systematic and random errors have mostly reduced: for about 0,1 - 0,5 mm. Consequently narrower PTV-CTV margin is neeeded, to be specific - 3,3 mm in AP, 2,6 mm in SI and 3,2 mm in ML direction. Discussion and conclusion: Conclusion, based on the results, shows that imobilization with individual headrest reflects in minor interfractional movements and lower frequency of isocenter correction. Statistically significant differences (p<10-3) were found between individual and standard headrests. An extenstion, no greater than 4 mm, of clinical target volume margin ensures that 90% of patients will recieve a minimum cumulative dose greater than 95% of the prescribed dose
Vpliv telesne sestave, stanja prehranjenosti in kaheksije na bioelektrični impedančni fazni kot pri bolnikih z rakom glave in vratu
No full textEfficiency of electrochemotherapy in treatment of head and neck cancer
No full textZnanstveno 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
Cellular resistance mechanisms in pre-irradiated tumours to electrochemotherapy with cisplatin or bleomycin
No full textIzhodišča: 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 bleomicinin (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 bleomycinand (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
Impact of delay in radiotherapy of head and neck tumors on treatment outcome
No full textBolniki s ploščatoceličnim karcinomom glave in vratu (PCKGV) čakajo na obravnavo zaradi kasnitev, ki se lahko zgodijo na ravni bolnika ali ob njegovem vstopu v zdravstveni sistem. Vsako čakanje v procesu obravnave teh bolnikov nosi tveganje za slabši izid zdravljenja. Z namenom, da bi določili še sprejemljivi časovni interval do začetka zdravljenja z radioterapijo (RT) ali sočasno kemoradioterapijo (KTRT), smo v kohorti slovenskih bolnikov s PCKGV, analizirali vpliv trajanja simptomov, dignostičnih postopkov, trajanja čakanja na pričetek RT ter kinetike rasti tumorjev na lokalno, regionalno, oz. sistemsko ponovitev bolezni in preživetje bolnikov.Introduction. Patients with squamous cell carcinoma of the head and neck (PCCHN) are awaiting treatment due to delays that may occur at the patient’s level or upon her/his entry into the health care system. Any waiting in the process of treating these patients carries the risk of a poorer treatment outcome. In order to determine an acceptable time interval until the start of treatment with radiotherapy (RT) or concurrent chemoradiotherapy (ChT-RT), we analyzed in a cohort of Slovenian patients with SCCHN the effect of the duration of symptoms, diagnostic procedures, the duration of waiting for the onset of RT and the kinetics of tumor growth to the local, regional, and systemic disease recurrence and patient survival
Effects of electrochemotherapy and ionizing radiation on immunologically important modifications in tumor cells
No full textZnanstveno izhodišče: Za zdravljenje tumorjev se uporabljajo različne ablativne terapije, med njimi radioterapija (RT) in elektrokemoterapija (EKT). Pri RT tumorske celice umirajo zaradi učinkov ionizirajočega sevanja (IR), pri EKT, ki združuje citostatike in elektroporacijo tkiva, pa tumorske celice umirajo zaradi citotoksičnosti zdravila na mestu aplikacije električnih pulzov. Obe terapiji s svojim delovanjem povzročata poškodbe dednega materiala tumorskih celic (DNA), s čimer onemogočata njihovo nadaljnjo delitev, in sprožita aktivacijo imunskega sistema ter povzročita različne vrste celične smrti. Mednje uvrščamo imunogeno celično smrt (ICD), apoptozo, nekrozo in avtofagijo. ICD predstavlja način umiranja celic, kjer je spodbujen pridobljen imunski odziv proti neoantigenom. Molekule, vpletene v ICD, so: na celični membrani izpostavljen kalretikulin (CRT), izven celice sproščen protein HMGB1 ter adenozin trifosfat (ATP). Poleg tega k imunskemu (ne)odzivu prispevajo tudi druge, imunološko pomembne spremembe v tumorskih celicah, ki vključujejo molekule MHC I in II ter PD-L1 in CD40. Dokazano je, da lahko RT izzove aktivacijo gostiteljevega imunskega sistema in sproži ICD, kar se odraža v imunskem spominu ter včasih sistemskih protitumorskih učinkih, ni pa znano, katere doze in režimi najučinkoviteje aktivirajo imunski sistem pri različnih vrstah tumorskih modelov. Učinkovitost EKT je sicer odvisna od imunogenosti tumorjev in njihove intrinzične občutljivosti na citostatike. Za optimizacijo aktivacije imunskega odziva z EKT je zato treba določiti, kateri citostatik, koncentracija in časovni okvir po terapiji izzovejo največ imunološko pomembnih sprememb v tumorskih celicah. Namen doktorske naloge je bil tako ugotoviti, kateri citostatik, ki se uporablja v EKT, ter v kakšni koncentraciji in v katerem časovnem okviru po terapiji v največji meri izzove imunološko pomembne spremembe v tumorskih celicah in vitro ter te primerjati s spremembami po IR.
Metode: V prvem delovnem sklopu doktorske naloge smo določili občutljivost mišjih tumorskih celičnih linij B16-F10 (melanom), 4T1 (karcinom dojke) in CT26 (rak debelega črevesa) in vitro na EKT z različnimi citostatiki in na IR. Najprej smo določili elektropermeabilnost celičnih linij, tako da smo suspenzijskim celicam dodali propidijev jodid, nato pa to suspenzijo napipetirali med dve ploščati elektrodi (z razdaljo med elektrodama 2,4 mm) ter jim dovedli električne pulze (8 pulzov, 100 µs, 1 Hz), ki smo jim spreminjali napetost na razdalji. V nadaljevanju smo določili preživetje celičnih linij po EKT s cisplatinom (CDDP), oksaliplatinom (OXA) in bleomicinom (BLM) in IR s testom klonogenosti. Z generatorjem električnih pulzov Jouan GHT beta smo celicam dovedli električne pulze, ki se uporabljajo pri EKT v kliniki (8 pulzov, 1300 V/cm, 100 µs, 1 Hz), nato pa celice v različnem številu nasadili na plošče. Za določitev radioobčutljivosti smo celice v različnem številu najprej nasadili na plošče in jih po treh urah obsevali z različnimi dozami IR. Za vsak citostatik smo iz krivulj preživetja določili inhibitorne koncentracije, z linearno-kvadratnim modelom pa smo izračunali inhibitorne doze IC30, IC50, IC70. V drugem delovnem sklopu smo celice in vitro tretirali z inhibitornimi dozami, nato pa ugotavljali izpostavljenost CRT na celični membrani s pretočnim citometrom. Sproščanje HMGB1 smo določili s testom ELISA, sproščanje ATP pa s testom za določanje luminiscence. Za določitev apoptoze in nekroze smo uporabili dve metodi. Pri prvi metodi smo celične linije in vitro tretirali z inhibitornimi dozami, nato pa za določanje uporabili pretočni citometer. Pri drugi metodi pa smo celice nasadili na ploščice, jih tretirali z inhibitornimi dozami, dodali fluorescentna reagenta ter s slikanjem v časovni seriji spremljali sproti obarvane apoptotične in nekrotične celice. Za določitev avtofagije smo celične linije in vitro tretirali z IC50, nato pa jih nasadili na plošče in inkubirali 24 ur. Za analizo smo uporabili test za določitev avtofagije, slike preparatov pa smo zajemali z mikroskopom. V zadnjem, tretjem delovnem sklopu smo določili spremembe v prisotnosti imunološko pomembnih membranskih označevalcev po obeh terapijah. S pretočnim citometrom smo preverili izražanje celičnih označevalcev MHC I, MHC II, PD-L1 ter CD40 v različnih časovnih točkah po tretiranju z inhibitornimi dozami in vitro.
Rezultati: V prvem delovnem sklopu smo ugotovili, da so vse tri celične linije (B16-F10, 4T1, CT26) primerljivo permeabilne pri vseh testiranih napetostih ter so bile vse tri enako občutljive na CDDP in BLM ter IR. V drugem sklopu smo pokazali, da celice po EKT večinoma umrejo takoj, medtem ko celice po IR poskušajo nastale poškodbe DNA najprej popraviti in je celična smrt pravzaprav posledica neuspešnega popravila. V splošnem je EKT imela ugodnejše učinke na nastanek ICD tudi pri manj imunogenih celičnih linijah v primerjavi z IR, saj je povzročila ICD pri več linijah ter pri različnih koncentracijah, medtem ko se je pri IR to zgodilo samo pri celični liniji CT26. Nadalje smo določali pojav apoptoze in nekroze po obeh terapijah, kjer smo opazili, da se po EKT pojavita dva neodvisna vrhova, v katerih celice umirajo. Prvi se pojavi takoj po EKT, drugi pa 24 do 48 ur po EKT s prevladujočim nekrotičnim načinom umiranja, ne glede na vrsto citostatika ali celično linijo. Nasprotno pa smo opazili po IR, kjer so celice umirale šele v poznejših časovnih točkah, kjer je prav tako prevladovala nekroza. Pri vseh treh celičnih linijah se je število mrtvih celic višalo z višanjem doze ter daljšanjem časa po IR. V nadaljnjih poskusih smo povišanje avtofagije opazili samo pri celični liniji 4T1 po EKT s CDDP in BLM, nasprotno pa smo ugotovili, da se je avtofagija po IR znižala pri celični liniji B16-F10. Pri preučevanju sprememb v prisotnosti MHC I, MHC II, PD-L1 ter CD40 smo pokazali, da je EKT z vsemi tremi citostatiki sprožila povišanje izražanja MHC I in PD-L1 pri vseh treh celičnih linijah, izražanje MHC II pa se je znižalo pri vseh citostatikih, razen pri celični liniji B16-F10, kjer se je izražanje povišalo. V nadaljevanju smo pokazali, da molekulo CD40 izraža samo celična linija 4T1, pri kateri se je izražanje CD40 povišalo po EKT z vsemi citostatiki, razen pri koncentracijah IC30 in IC50 BLM. Tudi IR je povišalo izražanje molekul MHC I pri vseh treh celičnih linijah ter vseh dozah sevanja, razen pri celični liniji 4T1 pri dozi IC30. Tudi izražanje PD-L1 se je povišalo pri vseh treh linijah in dozah, pri 4T1 pa tudi CD40. Pri molekuli MHC II smo največ sprememb ugotovili pri celični liniji B16-F10.
Zaključki: V raziskavi smo dokazali, da EKT s klinično pomembnimi citostatiki CDDP, OXA in BLM izzove različne imunološko pomembne spremembe v tumorskih celicah in vitro, odvisno od vrste citostatika, njegove koncentracije ter tumorskega modela. Pokazali smo tudi, da IR povzroči primerljive spremembe v izražanju celičnih površinskih označevalcev MHC I, MHC II, PD-L1 in CD40, vendar to ni časovno usklajeno s spremembami po EKT. EKT je sprožila največ sprememb v izražanju z ICD povezanih molekul DAMP (CRT, HMGB1, ATP) pri manj imunogeni celični liniji 4T1, IR pa je sprožilo največ sprememb v izražanju z ICD povezanih molekul DAMP pri najbolj imunogeni celični liniji CT26. EKT s klinično pomembnimi citostatiki CDDP, OXA in BLM zato lahko uvrstimo na seznam terapij, ki sprožijo ICD in vitro. Z našimi rezultati smo omogočili mehanističen vpogled v to, kako izkoristiti potencial ICD, da se sproži sistemski protitumorski imunski odziv, še posebej ko EKT kombiniramo z imunoterapijo.Scientific background: Various ablative techniques are used to treat tumors, including radiotherapy (RT) and electrochemotherapy (ECT). With RT, the tumor cells die due to the effect of ionizing radiation (IR), while with ECT, as a result of a combination of chemotherapeutic agents and the electroporation of tissues, the tumor cells die at the site where the electrical pulse is applied due to the cytotoxic effect of the drug. Both therapies damage the genetic material of the tumor cells (DNA) and thus prevent their further proliferation, trigger the activation of the immune system and cause various types of cell death. These include immunogenic cell death (ICD), apoptosis, necrosis, and autophagy. ICD is a form of cell death in which the adaptive immune system is stimulated against tumor neoantigens. The molecules involved in ICD are: calreticulin exposed on the cell membrane (CRT), extracellularly released HMGB1, and adenosine triphosphate (ATP). In addition to these molecules, the immune (non-)response is influenced by other immunologically important changes in tumor cells, including the molecules MHC I and II, PD-L1, and CD40. RT has been shown to stimulate activation of the host immune system and trigger ICD, resulting in immune memory and sometimes systemic antitumor effects. However, it is not yet known which doses and procedures activate the immune system most effectively in different tumor models. The efficacy of ECT depends on the immunogenicity of the tumors and their intrinsic sensitivity to chemotherapeutic agents. Therefore, the type of chemotherapeutic agent, its concentration, and the time frame after therapy that induces the most immunological changes in tumor cells must be determined to optimize the activation of the immune response by ECT. The aim of this dissertation was to determine which chemotherapeutic agent used in ECT induces the most important immunological changes in tumor cells in vitro, at which concentration, and in which time frame after therapy, and to compare these with the changes after IR.
Methods: In the first part of the dissertation, we investigated the sensitivity of the murine tumor cell lines B16-F10 (melanoma), 4T1 (mammary carcinoma), and CT26 (colorectal carcinoma) in vitro to ECT with different chemotherapeutic agents and to IR. First, we determined the electropermeability of the cell lines by adding propidium iodide to the suspension cells. The suspension was pipetted between two stainless steel plate electrodes (2.4 mm between the electrodes) and electroporated with pulses (8 pulses, 100 µs duration, 1 Hz) with different amplitude–to–distance ratios. In the next part of the study, we determined the survival of the cell lines after ECT using cisplatin (CDDP), oxaliplatin (OXA), and bleomycin (BLM), and after IR with a clonogenic assay. The Jouan GHT beta electrical pulse generator was used to deliver electrical pulses as used in clinical ECT (8 pulses, 1300 V/cm, 100 µs duration, 1 Hz), and cells were seeded in plates at different densities. To determine the radiosensitivity, the cells were seeded in plates at different densities and irradiated with different doses after three hours. The inhibitory concentrations were determined for each chemotherapeutic agent using the survival curves, and the inhibitory doses IC30, IC50, IC70 were calculated using a linear-quadratic model. In the second part, cells were treated in vitro with inhibitory doses, and then the exposure of CRT was determined by flow cytometry. HMGB1 release was determined by ELISA assay, and ATP release by luminescence assay. Two methods were used to determine apoptosis and necrosis. In the first method, the cell lines were treated in vitro with inhibitory doses, and then flow cytometry was used for determination. In the second method, cells were seeded on plates, treated with inhibitory doses, then fluorescent reagents were added and cells were monitored with time-lapse imaging. To determine autophagy, the cell lines were treated in vitro with IC50, then seeded on plates and incubated for 24 hours. The assay to determine autophagy was used for analysis, and images of the samples were taken with a microscope. In the last part, we determined the changes in the presence of immunologically important membrane markers after both therapies. The expression of the cell markers MHC I, MHC II, PD-L1, and CD40 was determined in vitro at different time points after treatment with inhibitory doses.
Results: In the first part, we found that all three cell lines (B16-F10, 4T1, CT26) were comparably permeable at all tested voltage-to-distance ratios and equally sensitive to CDDP, BLM, and IR. In the second part, we showed that cells usually die immediately after ECT, whereas after IR they try to repair the DNA damage caused, and that cell death is actually the result of unsuccessful repair. In general, ECT had more favorable effects on the development of ICD compared to IR, even in less immunogenic cell lines, as it caused ICD in more lines and at different concentrations. In contrast, IR caused ICD only in CT26. Furthermore, we determined the development of apoptosis and necrosis after both therapies and showed that two separate peaks of cell death occur after ECT. The first occurs immediately after ECT and the second 24 to 48 hours after ECT, with necrosis being the main modality of death, regardless of the type of chemotherapeutic agent or cell line. In contrast, cells died at later time points after IR, but again necrosis was the main modality. The number of dying cells increased in proportion to both the dose and the duration following IR. In further experiments, we found the increase in autophagy only in the 4T1 after ECT with CDDP and BLM. In contrast, we found a decrease in autophagy in B16-F10 after IR. When examining the changes in the expression of MHC I, MHC II, PD-L1, and CD40, we were able to show that all chemotherapeutic agents caused an increase in the expression of MHC I and PD-L1 in all cell lines after ECT. The expression of MHC II decreased with all chemotherapeutics except for the B16-F10 cell line, where expression increased. Next, we showed that the CD40 molecule is expressed only by the 4T1 cell line and that its expression increased after ECT with all chemotherapeutics, except IC30 and IC50 of BLM. Furthermore, the expression of MHC I increased in all cell lines and after all doses used after IR, except in 4T1 at dose IC30. The expression of PD-L1 also increased in all cell lines and doses, and the expression of CD40 also increased in 4T1. Most changes in the MHC II molecule were observed in the B16-F10 cell line.
Conclusions: With this study, we showed that ECT, together with the clinically relevant chemotherapeutics CDDP, OXA, and BLM, induces different immunologically important changes in tumor cells in vitro, depending on the type of chemotherapeutic agent, its concentration, and the tumor model. In addition, IR induces comparable changes in the expression of the cell surface markers MHC I, MHC II, PD-L1, and CD40, which, however, are not correlated in time with the changes after ECT. ECT caused the most changes in the expression of the DAMP molecules (CRT, HMGB1, ATP), which correlated with ICD in the less immunogenic cell line 4T1. IR caused the most changes in the expression of DAMP in the most immunogenic cell line CT26. ECT, together with the clinically relevant chemotherapeutics CDDP, OXA, and BLM, can therefore be included in the list of therapies that induce ICD in vitro. Our results provide mechanistic insights into how the potential of ICDs to trigger a systemic anti-tumor immune response can be exploited, especially when ECT is combined with immunotherapy
review Role of radiotherapy in melanoma management
No full textDisclosure: No potential conflicts of interest were disclosed. Background. In melanoma, radiotherapy has generally been considered as a palliative treatment option indicated only for advanced cases or disseminated disease. In the 70s of the previous century, the technological advances in radiotherapy, linked to rapid development of computer sciences, resulted in restored interest for radiotherapy in melanoma management. Although a fundamental lack of well designed prospective and/or randomized clini-cal trials critically influenced the integration of radiotherapy into treatment strategies in melanoma, radiotherapy was recently recognized as an indispensable part in the multidisciplinary management of patients with melanoma. Altogether, approximately 23 % of melanoma patients should receive at least one course of radiotherapy during the course of the disease. In this review, radiobiological properties of melanoma that govern the decisions for the frac-tionation patterns used in the treatment of this disease are described. Moreover, the indications for irradiation and the results of pertinent clinical studies from the literature, creating a rationale for the use of radiotherapy in the manage-ment of this disease, are reviewed and a brief description of radiotherapy techniques is given. Conclusions. Basic treatment modality in melanoma is surgery. However, whenever surgery is not radical or there are adverse prognostic factors identified on histopathological examination of resected tissue specimen, it needs to be supplemented. Also, in patients with unresectable disease or in those not being suitable for major surgery or wh
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