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Rak debelega črevesa in danke (RDČD) je med najpogostejšimi rakavimi obolenji. Večina RDČD se pojavi sporadično, poznamo pet stopenj, od zdrave črevesne sluznice, adenomov do napredovalega karcinoma z zasevki. Pomembno vlogo v razvoju RDČD imajo tumorske celice z lastnostmi matičnih celic (MC). Predpostavljamo, da so razlike v izražanju izbranih genov povezane z MC, v razvoju RDČD in da izbrane miRNA regulirajo izbrane gene. Z bioinformatskimi orodji smo analizirali prosto dostopne podatke izražanja RNA, pridobljene s sekvenciranjem in mikromrežami za vzorce adenomov, RDČD in pripadajoče črevesne sluznice. Za nadaljnjo validacijo s kvantitativno verižno reakcijo s polimerazo smo izbrali gene, povezane z MC (ANLN, CDK1, ECT2 L1TD1, PDGFD, SLITRK6, ST6GALNAC1, TCEA3, TNC in TNXB) in napovedane izbrane potencialno regulatorne mikro RNA (miRNA). Za validacijo izražanja smo uporabili tkivne vzorce bolnikov z adenomi, adenomi z zgodnjim RDČD, RDČD brez zasevanja in z zasevanjem. Funkcijo izbranega proteina smo opredelili z utišanjem na celičnih linijah PC3 in HT-29. Pridobili smo dokaze, da je spremenjeno izražanje genov ANLN, ECT2, TCEA3 in TNC potencialno povezano z maligno transformacijo, z zasevanjem pa genov ANLN, CDK1, ECT2, L1TD1, SLITRK6, ST6GALNAC1 in TCEA3. Izbranih genov nismo uspeli povezati z predpostavljenimi potencialno regulatornimi miRNA. Colorectal cancer (CRC) is among the most common cancers. Most CRCs are sporadic, CRC develops in five stages, from healthy mucosa to adenoma and late stage carcinoma. An important role in CRC development could be associated with cancer cells with stem cell properties (SC). We hypothesize that the differences in expression of selected genes are associated with SC in CRC development, and that selected miRNAs regulate these genes. Using bioinformatics tools we analyzed publicly available RNA data obtained with sequencing and microarrays for samples of adenomas, CRC and corresponding colon mucosa. For further validation, using quantitative polymerase chain reaction, we chose genes associated with SC (ANLN, CDK1, ECT2, L1TD1, PDGFD, SLITRK6, ST6GALNAC1, TCEA3, TNC and TNXB) and selected, predicted potentially regulatory microRNAs (miRNAs). For validation of expression we used tissue samples from patients with adenomas, adenomas with early carcinoma, CRC without and with metastases. The function of the selected protein was characterized by silencing in PC3 and HT-29 cell lines. We found evidence that differential expression of genes ANLN, ECT2, TCEA3 and TNC is potentially associated with malignant transformation, while differential expression of genes ANLN, CDK1, ECT2, L1TD1, SLITRK6, ST6GALNAC1 and TCEA3 is potentially associated with metastasizing. We found no significant association between selected potentially regulatory miRNAs and selected genes.

Pri resektabilnem raku kolona je indicirano kirurško zdravljenje. Namen tega zdravljenja je radikalna odstranitev tumorja z žilnim pecljem po principu kompletne mezokolične ekscizije. Pri desni hemikolektomiji, ki je standardna operacija pri raku slepiča, cekuma in ascendentnega kolona se zadnje desetletje izpostavlja D3 limfadenektomija. Nekatere raziskave so pokazale, da D3 v primerjavi z D2 limfadenektomijo omogočala daljše preživetje brez bolezni. D3 limfadenektomijo so nekatere azijske države že uvedle med priporočila za kirurško zdravljenje raka kolona. Kljub temu ni dovolj trdnih dokazov, da bi podobne smernice sprejeli tudi drugod po svetu. Surgical treatment is indicated for resectable colon cancer. The aim of this treatment is the radical removal of the tumor with a vascular stalk according to the principle of complete mesocolic excision. For right hemicolectomy, which is a standard operation for cancer of the appendix, cecum and ascending colon, D3 lymphadenectomy has been performed in the last decade. Some studies have shown that D3 compared to D2 lymphadenectomy allowed longer disease-free survival. Some Asian countries have already introduced D3 lymphadenectomy among the recommendations for the surgical treatment of colon cancer. Nevertheless, there is not enough solid evidence to adopt similar guidelines elsewhere in the world.

Celostna rehabilitacija vključuje aktivne postopke, s katerimi onkološkim bolnikom omogočimo najboljše telesno, duševno in socialno delovanje od diagnoze dalje. Številne klinične raziskave kažejo, da onkološko zdravljenje povzroča številne posledice, kar bolnikom prinaša mnogotere težave, zaradi katerih se njihovo življenje korenito spremeni. Celostna rehabilitacija za onkološke bolnike se danes v Sloveniji ne izvaja na ravni države. Bolniki so deležni rehabilitacije po onkološkem zdravljenju sporadično in v neenakomernem obsegu v posameznih ustanovah, kjer se zdravijo bolniki z rakom in v zdravstvenih domovih. V okviru projekta Organizirane celostne rehabilitacije bolnikov z rakom debelega črevesa in danke bomo poiskušali ugotoviti, ali je kakovost življenja bolnikov z rakom debelega črevesa in danke, ki so vključenih v interventno skupino boljša. S primerjavo interventne in kontrolne skupine bomo ugotovili tudi, kako je intervencija vplivala na trajanje njihove bolniške odsotnosti in na invalidsko upokojevanje. Na osnovi analize rezultatov bomo pripravili načrtovano oceno potreb po celostni rehabilitaciji za rak debelega črevesa in danke, ki bo podlaga za načrtovanje resursov na državni ravni. Comprehensive rehabilitation includes active procedures that enable oncology patients to have the best physical, mental and social functioning from diagnosis onwards. Numerous clinical studies show that oncological treatment causes many consequences, which brings many problems to patients, due to which their lives change radically. Comprehensive rehabilitation for oncology patients is currently not carried out at the national level in Slovenia. Patients receive rehabilitation after oncological treatment sporadically and to an uneven extent in individual institutions where cancer patients are treated and in medical centers. Within the framework of the project Organized comprehensive rehabilitation of patients with colon and rectal cancer, we will try to determine whether the quality of life of patients with colon and rectal cancer, who are included in the intervention group, is better. By comparing the intervention and control groups, we will also determine how the intervention affected the duration of their sick leave and disability retirement. Based on the analysis of the results, we will prepare a planned assessment of the needs for comprehensive rehabilitation for colon and rectal cancer, which will be the basis for resource planning at the national level.

V prispevku je prikazan klinični primer bolnika z inoperabilnim recidivom karcinoma danke, ki je bil zdravljen z visokodoznim obsevanjem z ogljikovimi ioni v protonskem centru v tujini. Obsevanja s protoni ali težkimi ioni v Sloveniji zaenkrat še ne moremo zagotoviti, zato moramo vse bolnike za katere presodimo, da bi imeli od tovrstnega zdravljenja korist, napotiti na zdravljenje v enega od protonskih centrov v tujini. V primerjavi z obsevanjem s fotoni, lahko zaradi specifičnih fizikalnih lastnosti protonskih delcev in težkih ionov, pri obsevanju z njimi bistveno zmanjšamo volumne v obsevalna polja zajetih zdravih tkiv, v določenih kliničnih situacijah pa tumorje tudi obsevamo z višjimi dozami. The article presents a clinical case of a patient with inoperable rectal carcinoma relapse who was treated with high-dose carbon ion irradiation at a proton center abroad. Irradiation with protons or heavy ions cannot be guaranteed in Slovenia for the time being, so we must refer all patients who we judge would benefit from this type of treatment to one of the proton centers abroad. Compared to irradiation with photons, due to the specific physical properties of proton particles and heavy ions, when irradiating with them, we can significantly reduce the volumes in the radiation fields of covered healthy tissues, and in certain clinical situations tumors can also be irradiated with higher doses.

Rak debelega črevesa in danke lahko napreduje v oligometastatsko bolezen. Napredki v zgodnjem odkrivanju in intenzivnem sistemskem zdravljenju omogočajo čedalje boljše odgovore na zdravljenje in dolgoročno sistemsko kontrolo bolezni. Pojavlja se vprašanje dolgoročnega vodenja oligometastatskih bolnikov. Kirurška resekcija zasevkov lahko pripomore k izboljšanem preživetju, omogoča kurativnost posega ali podaljša interval brez sistemskega zdravljenja, je pa pogosto omejena z anatomijo, invazivnostjo in morbiditeto takšnega pristopa. Stereotaktično obsevanje (SBRT) zasevkov omogoča zelo učinkovito in natančno zdravljenje zasevkov v nizkem številu obsevalnih odmerkov in s tem zelo dobro in dolgoročno lokalno kontrolo z minimalno invazivnostjo in morbidnostjo. Ta prikaz primera prikazuje zgodbo bolnika, zdravljenega na Onkološkem inštitutu v Ljubljani. Colorectal cancer can progress to oligometastatic disease. Advances in early detection and intensive systemic treatment enable increasingly better responses to treatment and long-term systemic control of the disease. The question of long-term management of oligometastatic patients arises. Surgical resection of implants may contribute to improved survival, enable curative intervention or prolong the systemic treatment-free interval, but is often limited by the anatomy, invasiveness and morbidity of such an approach. Stereotactic radiation (SRS) of metastases enables very effective and precise treatment with a low number of radiation doses and thus very good and long-term local control with minimal invasiveness and morbidity. This case report shows the story of a patient treated at the Oncology Institute in Ljubljana.

V zadnjih treh desetletjih se incidenca raka debelega črevesa in danke med mlajšimi v razvitem svetu povečuje. Predvidevajo, da bo v naslednjih desetih letih vsak deseti bolnik z zrakom debelega črevesa in vsak četrti z rakom danke mlajši od 50 let. Vzrokov za zbolevanje vse mlajših generacij še ne poznamo. Bolezen se pri mlajših pogosteje odkrije pozno, v napredovalem stadiju. Pogosto bolniki, včasih tudi zdravstveni izvajalci, resnosti opozorilnih simptomov in znakov dlje časa ne prepoznajo. Osveščanje strokovne in laične javnosti o pojavljanju te bolezni med mlajšimi je zato ena od prioritet pri izboljšanju obravnave in preživetja bolnikov. V pripravi so tudi smernice, ki bodo zaobjele široko področje diagnostike, genetske obravnave, zdravljenja in širše oskrbe mlajših bolnikov.

Incidenca in umrljivost pri raku debelega črevesa in danke pri mladih v zadnjih desetletjih naraščata. Vsak deseti z diagnozo raka debelega črevesa in danke je mlajši od 50 let. Spremenjene prehranske navade in življenski slog sta glede na zadnje študije pomembna dejavnika tveganja predvsem zaradi vpliva na gastroinestinalni mikrobiom, ki se posredno preko številnih interakcij vpleta v posameznikov imunski odgovor. Delež rakov, ki so odkriti v napredovalem stadiju III ali IV je pri mlajših od 50 let višji. V nadaljevanju predstavljamo dva klinična primera s poudarkom na času od pojava simptomatike do postavitve diagnoze raka debelega črevesa in danke.

Rak debelega črevesa in danke (RDČD) v svetovnem pogledu predstavlja drugi najpogostejši vzrok smrti in po incidenci sodi na tretje mesto med vsemi raki. V zadnjih 10-15. letih smo lahko priča velikim napredkom v sistemskem zdravljenju RDČD in to je poleg uspešnih preventivnih programih glavni razlog da v tem trenutnku RDČD spada med bolezni z dobrim preživetjem. V mednarodni raziskavi Concord-3 je bilo ugotovjeno, da petletno čisto preživetje bolnikov v več kot polovici evropskih držav že presega 60 %. Najpomembnejši prognostični dejavnik je stadij ob diagnozi, saj je pet-letno čisto preživetje bolnikov z omejenim stadijem že več kot 90 %, medtem ko je preživetje tistih z razsejano boleznijo pa komaj 9 %. Pri 50% bolnikov z N+ boleznijo ob ugotovitvi bolezni v poteku njihove bolezni le-ta postane metastatska. V nasprotju z splošnim prepričanjem je tudi RDČD imunogena maligna bolezen, vendar le v primeru ko gre za MSI-H (mikrosatelitno nestabilno) obliko bolezni. Tovrstni tumorji imajo namreč preko 1500 mutacij na tumor, kar je napovedni dejavnik za učinkovitost zdravljenja z imunoterapijo (IT). Tako ni presenečenje, da je IT lahko zelo učinkovita možnost zdravljenja pri bolnikih z metastatskim RDČD in je tudi že del mednarodnih in slovenskih smernic zdravljenja RDČD.

Colorectal cancer (CRC) is still one of the most common types of cancer in the world, and the gut microbiome plays an important role in its development. The microbiome is involved in the carcinogenesis, formation and progression of CRC as well as its response to different systemic therapies. The composition of bacterial strains and the influence of geography, race, sex, and diet on the composition of the microbiome serve as important information for screening, early detection and prediction of the treatment outcome of CRC. Microbiome modulation is one of the most prospective new strategies in medicine to improve the health of individuals. Therefore, future research and clinical trials on the gut microbiome in oncology as well as in the treatment of CRC patients are warranted to determine the efficacy of systemic treatments for CRC, minimize adverse effects and increase survival rates. Nasl. z nasl. zaslona. Opis vira z dne 13. 12. 2021. Bibliografija na koncu prispevka. Abstract.

Znanstveno izhodišče: Avtologne tumorske vakcine so tip terapevtskih vakcin, s katerimi želimo spodbuditi bolnikov imunski odziv za boj proti rakavemu obolenju. Sestavljene so iz gensko spremenjenih tumorskih celic, inaktiviranih z obsevanjem, ali iz tumorskega lizata. Nabor avtolognih tumorskih vakcin je majhen, med njimi pa še nobena ni bila odobrena za standardno zdravljenje. Namen doktorske raziskave je bil priprava in testiranje nove avtologne tumorske vakcine, pripravljene iz mrtvih tumorskih celic, z adjuvantnim genskim elektroprenosom plazmidne DNA z zapisom za interlevkin-12 (GEP IL-12) in vivo. Učinkovitost vakcine smo testirali v kombinaciji z lokalno radioterapijo, s katero smo želeli izpostaviti tumorske antigene, in zaviralci imunskih kontrolnih točk (ZIKT), protitelesi anti PD-1 in anti CTLA-4, s katerima smo želeli sprositi morebitno zavrtje imunskega sistema. Metode: Za raziskave smo uporabili mišje tumorske celice B16-F10 in CT26. Poskusi in vivo so potekali na miših C57Bl/6NCrl in Balb/cAnNCrl, ki so singenske izbranima tumorskima modeloma. Protitumorski učinek terapij smo spremljali z merjenjem velikosti tumorjev. Namen prvega dela raziskav je bil razvoj učinkovite tumorske vakcine B16-F10 in CT26. Pilotno vakcino smo pripravili z obsevanjem celic B16-F10 z dozo 25 Gy in jo testirali in vivo v kombinaciji z obsevanjem tumorjev. Pilotno vakcinacijo smo izvedli tako, da smo v podkožje miši, oddaljeno od tumorja, injicirali mrtve tumorske celice in izvedli GEP IL-12 okoli injiciranih mrtvih tumorskih celic (2,82 Hz, 170 V/cm, 150 ms). V nadaljevanju smo glede na rezultate histološke analize mesta pilotne vakcinacije izboljšali postopek priprave vakcine. Natančneje, izboljšano vakcino B16-F10 smo pripravili z obsevanjem 3 × 5 Gy in nato 30 Gy, izboljšano vakcino CT26 pa z obsevanjem 3 × 5 Gy in nato 2 × 30 Gy. Izboljšano vakcinacijo smo izvedli tako, da smo v podkožje miši, oddaljeno od tumorja, injicirali mešanico mrtvih tumorskih celic in plazmida IL-12 in izvedli GEP IL-12 v mešanici (5,64 Hz, 170 V/cm, 150 ms). Za določitev doprinosa vakcinacije k protitumorskemu učinku obsevanja (15 Gy) in vpliva vakcinacije na izrast tumorjev smo testirali dva odmerka vakcine (0,5 in 1 mg). Namen drugega dela raziskave je bila primerjava različnih obsevalnih in vakcinacijskih režimov. Najprej smo primerjali tri različne obsevalne režime: 1 × 5, 3 × 5 in 5 × 5 Gy v kombinaciji z vakcinacijo (1 mg) ob prvi frakciji obsevanja. Nato smo primerjali tri vakcinacijske režime: enkratno in večkratno vakcinacijo sočasno z obsevanjem ter večkratno vakcinacijo, ki smo jo začeli pred prvo dozo obsevanja. Namen tretjega dela raziskave je bilo ovrednotenje lokalnega in sistemskega imunskega odziva po kombinirani terapiji z izbranim obsevalnim in vakcinacijskim režimom pri obeh tumorskih modelih. Za imunohistokemijsko analizo smo odvzeli vzorce kože na mestu vakcinacije in tumorje, pri čemer smo označili imunske celice, pozitivne na grancim B, CD68 in FoxP3. Z analizo FluoroSpot smo analizirali prisotnost tumorsko specifičnih efektorskih imunskih celic, pozitivnih na grancim B in interferon ?, v bezgavkah. V četrtem delu raziskav smo ovrednotili doprinos ZIKT k vakcinaciji v kombinaciji z obsevanjem s protitumorsko učinkovitostjo ter histološko analizo tumorske nekroze in infiltracije imunskih celic v koži na mestu vakcinacije in v tumorjih. Rezultati: Po pilotni vakcinaciji smo na mestu vakcinacije opazili še žive tumorske celice ter večjo infiltracijo imunskih celic na mestu GEP kot na mestu injiciranih mrtvih tumorskih celic. Po izboljšanju postopka priprave vakcine na mestu izboljšane vakcinacije ni bilo več živih celic in opazili smo bolj enakomerno infiltracijo imunskih celic. Z enkratno izboljšano vakcinacijo smo doprinesli k protitumorskemu učinku obsevanja z dozo 15 Gy le pri tumorskem modelu B16-F10, ne pa tudi pri tumorskem modelu CT26. Po drugi stani pa smo z enkratno izboljšano vakcinacijo preprečili izrast do 56 % tumorjev CT26, ne pa tudi tumorjev B16-F10. Najučinkovitejši odmerek vakcine pri obeh tumorskih modelih je bil 1 mg. Najučinkovitejši obsevalni režim v kombinaciji z enkratno vakcinacijo pri obeh tumorskih modelih je bil 5 × 5 Gy. Najučinkovitejši vakcinacijski režim v kombinaciji s 5 × 5 Gy pri tumorskem modelu B16-F10 je bila enkratna vakcinacija, pri tumorskem modelu CT26 pa trikratna vakcinacija, izvedena sočasno z obsevanjem. Z vakcinacijo smo uspešno sprožili lokalni in sistemski imunski odziv, saj smo v kožo na mesto vakcine ter v tumor privabili makrofage, efektorske limfocite ter regulatorne limfocite. ZIKT niso doprinesli k protitumorskemu učinku vakcinacije v kombinaciji z obsevanjem ter niso povečali količine tumorske nekroze in infiltracije imunskih celic v tumor ali v kožo na mestu vakcinacije. Zaključki: Uspešno smo pripravili učinkovito avtologno tumorsko vakcino B16-F10 in CT26. S kombinirano terapijo z izbranimi obsevalnimi in vakcinacijskimi režimi smo pri obeh tumorskih modelih potrdili hipotezo, da vakcinacija doprinese k protitumorskemu učinku radioterapije. Prav tako smo pri obeh tumorskih modelih potrdili hipotezo, da z vakcinacijo izzovemo lokalni in sistemski imunski odziv. Zadnjo hipotezo smo ovrgli, saj smo dokazali, da ZIKT ne doprinesejo k protitumorskemu učinku vakcinacije v kombinaciji z obsevanjem. Rezultati doktorske raziskave bodo osnova za nadaljnji razvoj avtologne tumorske vakcine. Scientific Background: The goal of autologous tumor cell-based vaccines, a type of therapeutic vaccines, is to enable the patient's immune system to fight against cancer. These consist of genetically modified and with irradiation inactivated tumor cells or tumor cell lysates. The current repertoire of autologous tumor cell-based vaccines remains low. The objective of this doctoral research was to develop and test a novel autologous tumor cell-based vaccine, containing non-viable tumor cells, with adjuvant in vivo IL-12 GET. The efficacy of the vaccine was examined in combination with tumor irradiation therapy, the goal of which was to expose tumor antigens. We also examined the efficacy of vaccination in combination with tumor irradiation therapy and immune checkpoint inhibition with aniti-PD-1 and anti-CTLA-4 antibodies to release possible inhibition of the immune system. Materials and Methods: Murine B16-F10 and CT26 tumor cell lines were used during the doctoral research. In vivo experiments were performed in syngeneic C57Bl/6NCrl and Balb/cAnNCrl mice. The antitumor efficacy was determined through the measured tumor volume. In the first part of our research, we developed B16-F10 and CT26 autologous tumor cell-based vaccines. A pilot vaccine was prepared by irradiating B16-F10 cells with a dose of 25 Gy, which we then tested in vivo in combination with tumor irradiation. Pilot vaccination was performed with a subcutaneous injection of non-viable tumor cells and IL-12 GET was performed around the injection site (2.82 Hz, 170 V/cm, 150 ms). The vaccine preparation procedure was improved based on the histological analysis of the pilot vaccination site. The modified B16-F10 vaccine was prepared with 3 × 5 Gy and 30 Gy irradiation, while the modified CT26 vaccine was prepared with 3 × 5 Gy and 2 × 30 Gy irradiation. The modified vaccines were administered subcutaneously using a mixture of non-viable tumor cells and IL-12 plasmid followed by GET of IL-12 contained within the injected vaccine (5.64 Hz, 170 V/cm, 150 ms). Two vaccination doses (0.5 and 1 mg) were tested in combination with tumor irradiation (15 Gy) to determine the antitumor efficacy. The ability of vaccination (0.5 and 1 mg) to prevent tumor outgrowth was also examined. In the second part of our research, we compared different irradiation and vaccination regimens. First, we compared three irradiation regimens: 1 × 5, 3 × 5 and 5 × 5 Gy, in combination with concurrent single-dose vaccination (1 mg). Then we compared three vaccination regimens: single- and multiple-dose vaccination with concurrent irradiation as well as multiple-dose vaccination, which began before irradiation. In the third part of our research, we determined the local and systemic immune response to combination treatment with the chosen vaccination and irradiation regimens. To determine granzyme B, CD68 and FoxP3 positive immune cells, skin at the vaccination site and tumors were harvested for immunohistochemical analysis. To determine tumor specific granzyme B and interferon γ immune cells, lymph nodes were harvested for FluoroSpot analysis. In the final part of our research, we determined the antitumor efficacy of checkpoint inhibition in combination with vaccination and tumor irradiation. Histological analysis of tumor necrosis and immune cell infiltration in tumors and skin at the site of vaccination was also performed. Results: Viable tumor cells were observed at the pilot vaccination site. Additionally, greater infiltration of immune cells was observed at the site of IL-12 GET than at the site of injected non-viable tumor cells, whereas at the adjusted vaccination site, non-viable tumor cells and a more uniform distribution of immune cells was observed. Single-dose vaccination contributed to antitumor efficacy of irradiation (15 Gy) in the B16-F10 but not in the CT26 tumor model, whereas single-dose vaccination prevented outgrowth of up to 56 % CT26 tumors however, it did not prevent outgrowth of B16-F10 tumors. The most effective vaccine dose was 1 mg in both tumor models. The most effective irradiation regimen was 5 × 5 Gy with concurrent single-dose vaccination in both tumor models. The most effective vaccination regimens were single-dose vaccination and multiple-dose vaccination with concurrent irradiation (5 × 5 Gy) in the B16-F10 and the CT26 tumor models, respectively. By attracting macrophages, effector lymphocytes and regulatory lymphocytes, vaccination successfully elicited a local and systemic immune response. Finally, immune checkpoint inhibition did not contribute to antitumor efficacy of vaccination in combination with irradiation and it did not affect the amount of tumor necrosis or immune cell infiltration. Conclusions: We successfully developed effective B16-F10 and CT26 autologous tumor cell-based vaccines. By combining the chosen irradiation and vaccination regimens, we confirmed our hypothesis that vaccination contributes to irradiation in both tumor models. We also confirmed our next hypothesis that vaccination elicits a local and systemic immune response in both tumor models. We rejected the final hypothesis since immune checkpoint inhibition did not contribute to antitumor efficacy of vaccination in combination with irradiation. The results of this doctoral dissertation will be the foundation for further development of the autologous tumor cell-based vaccine.

Rak debelega črevesa in danke (RDČD) je posledica kopičenja morfoloških in (epi)genetskih sprememb. Kljub poznavanju histopatoloških značilnosti je pri mnogih lezijah postavitev pravilne diagnoze zaradi netipične histopatološke slike zahtevna. V pomoč bi lahko bili označevalci, specifični za različne stopnje razvoja RDČD. Opredelili smo vlogo šestih genov zunajceličnega matriksa (ECM), katerih izražanje se je glede na bioinformatske analize statistično značilno razlikovalo med adenomom in RDČD. V raziskavo smo vključili 105 biopsijskih tkivnih vzorcev 62 bolnikov, pri katerih so bili endoskopsko ali operativno odstranjeni adenom, adenom s psevdoinvazijo, maligniziran adenom ali RDČD. Eksperimentalno validacijo smo naredili na mRNA in proteinskem nivoju, analizo (epi)genetske regulacije pa na RNA in DNA nivoju. Na proteinskem in mRNA nivoju smo ugotovili, da je pri genih DCN, SPON2, SPARC in SPP1 izražanje premo-sorazmerno z malignostjo neoplazme. Na nivoju mRNA je bilo izražanje EPHA4 povišano pri adenomih s psevdoinvazijo, medtem ko je bilo izražanje FN1 v razvoju RDČD podobno kot v normalni sluznici. Ugotavljali smo tudi (epi)genetsko regulacijo genov, katerih izražanje se je spreminjalo v razvoju RDČD. Izražanje gena DCN je bilo soodvisno od miR-200c, medtem ko je bilo izražanje gena SPARC soodvisno od metilacijskega statusa promotorja. Naša analiza ni pokazala nobene statistično značilne povezave med izbranimi načini regulacije in izražanjem gena SPON2. Analiza (epi)genetske regulacije gena SPP1 pa je pokazala, da je izražanje gena SPP1 soodvisno tako od miR-146 kot od metilacijskega statusa promotorja in spremembe števila kopij gena. Z eksperimentalno validacijo izbranih genov in proteinov ECM smo ugotovili, da imajo pomembno vlogo v razvoju RDČD. Čeprav so vzorci njihovega izražanja preveč zapleteni, da bi jih lahko uporabili kot diagnostične označevalce pri vsakdanjem delu, pa prispevajo k boljšemu razumevanju sprememb ECM v razvoju RDČD, kar nam bo lahko v prihodnosti v pomoč pri odkrivanju novih označevalcev in načinov zdravljenja. Colorectal carcinoma (CRC) arises due to the accumulation of morphological and (epi)genetic changes. Despite well-defined histopathological features, there are many lesions with ambiguous histopathological features making diagnosing difficult. For this reason, markers specific for each stage of CRC development could be helpful. We experimentally validated six genes of the extracellular matrix (ECM), whose expression was identified in the bioinformatics analysis as significantly different between adenoma and CRC. Our study included 105 biopsy tissue samples from 62 patients who had endoscopically or surgically removed adenoma, adenoma with epithelial misplacement, malignant adenoma, or CRC. Experimental validation of expression was performed on mRNA and protein level and (epi)genetic regulation on DNA and RNA level. We showed that expression of DCN, SPON2, SPARC in SPP1 on mRNA and protein level increased with level of malignancy. Expression of EPHA4 was up-regulated in adenomas with epithelial misplacement, whereas expression of FN1 was similar to healthy mucosa throughout CRC development. Furthermore, we were interested in the (epi)genetic regulation of genes whose expression increased with level of malignancy. Expression of DCN was inversely proportional to miR-200c, while expression of SPARC was correlated to the methylation status of its promotor region. Our analysis did not show any significant associations between selected types of regulation and expression of SPON2. In contrast, expression of SPP1 was associated with the expression of miR-146a, the methylation status of the promotor region, and the copy number variation. Experimental validation of selected ECM genes and proteins provided further evidence of their important role in the development of CRC. Though the expression patterns of analysed genes and proteins are too complex to be used directly in diagnostic work as markers, they might contribute to a better understanding of ECM changes in CRC development and help in search for new marker(s) and treatment modalities in the future.

Rak debelega črevesa in danke (RDČD) predstavlja velik javnozdravstveni problem s pogosto slabo prognozo v primeru pozne diagnoze in terapije. Konvencionalne metode zdravljenja imajo številne slabosti in omejitve, kar spodbuja razvoj sodobnejših pristopov k zdravljenju, usmerjenih v ciljano dostavo učinkovin, kombinirano terapijo in teranostiko. Pomemben dejavnik tveganja za nastanek in napredovanje RDČD je vnetje v gastrointestinalnem traktu s posledično aktivacijo imunskih celic ter sproščanjem vnetnih citokinov. Z dostavo ustreznih protivnetnih učinkovin v črevesje lahko omilimo vnetje ter z njim povezane poškodbe sluznice in upočasnimo ali celo ustavimo progresijo RDČD, s hkratno dostavo vezalcev tumorsko specifičnih antigenov pa lahko dosežemo ciljano dostavo teh učinkovin. V sklopu raziskovalnega dela smo pripravili genske konstrukte in jih vstavili v plazmide pSDBA3b oziroma pNZDual za posamezno ali sočasno površinsko predstavitev dveh proteinov. V konstrukte smo vstavili gen za protein evazin (vezalec provnetnega IL-8) oziroma gen za protein afitin (vezalec tumorskega antigena EpCAM) ter zaporedje za peptidno oznako Cmyc ali Flag. Slednje so pomembno orodje v biokemiji, saj pripete na protein v kombinaciji s specifičnimi protitelesi omogočajo detekcijo, čiščenje in karakterizacijo rekombinantnih proteinov. Konstrukte smo izrazili v bakterijah L. lactis, ki so zaradi dolgoletne priznane varnosti in enostavne genske modifikacije primeren vektor za peroralno dostavo omenjenih terapevtskih proteinov. S pomočjo peptidnih oznak smo potrdili izražanje posameznih proteinov EVA-Cmyc, AFFI-Cmyc, EVA-Flag in AFFI-Flag oziroma njihovih kombinacij, dokazali njihovo prisotnost v celičnih lizatih in tudi na površini bakterij L. lactis, pri čemer je obseg površinske predstavitve variiral glede na vrsto proteina, uvedeno peptidno oznako in tudi glede na to, ali je bil protein izražen posamezno ali sočasno z drugim proteinom. Navsezadnje smo dokazali, da so bakterije s površinsko predstavljenimi evazini in afitini funkcionalne, torej sposobne vezave IL-8 in receptorja EpCAM. Zaradi splošnega neodobravanja GSO smo izražene proteine ločili od celic proizvajalk, jih nekovalentno pripeli na divje seve bakterij, ki niso bile gensko spremenjene in dokazali njihovo površinsko predstavitev. Celotno vrednotenje proteinov smo izvedli na osnovi peptidnih oznak in dokazali, da je njihova uporaba primeren, cenovno ugoden ter enostaven način za detekcijo in razlikovanje izraženih proteinov, kar je bil tudi osnovni namen magistrskega dela. Ugotovili smo, da oznake sicer sterično ovirajo vezavna mesta proteinov in vplivajo na njihovo funkcionalnost, a vendar prednosti njihove uporabe odtehtajo omenjene slabosti. Colorectal cancer (CRC) is a major public health problem with often poor prognosis in the case of a late diagnosis and therapy. Conventional methods for treatment have many disadvantages and limitations, which encourage the development of more modern approaches to treatment aimed at targeted active substance delivery, combination therapy and theranostics. An important risk factor for the development and progression of CRC is inflammation in the gastrointestinal tract with consequent activation of immune cells and the release of inflammatory cytokines. By delivering appropriate anti-inflammatory agents to the intestine, inflammation and associated mucosal damage can be alleviated and the progression of CRC can be slowed or even stopped. Concomitant delivery of tumor-specific antigen binders can achieve targeted delivery of these active ingredients. As part of the research work, we prepared gene constructs and inserted them into plasmids pSDBA3b or pNZDual for individual or simultaneous surface display of two proteins. The evasin protein gene (pro-inflammatory IL-8 binder) or the affitin protein gene (EpCAM tumor antigen binder) and the sequence for the peptide tag Cmyc or Flag were inserted into the constructs. The latter are an important tool in biochemistry, since they allow, while attached to a protein in combination with specific antibodies, the detection, purification, and characterization of recombinant proteins. The constructs were expressed in L. lactis bacteria, which are a suitable vectors for the oral delivery of the mentioned therapeutic proteins due to their long-term recognized safety and simple genetic modification. The expression of individual proteins EVA-Cmyc, AFFI-Cmyc, EVA-Flag, and AFFI-Flag or their combinations was confirmed by using peptide tags, their presence was proved in cell lysates and also on the surface of L. lactis bacteria, with the extent of surface presentation varying, depending on the type of protein, introduced peptide tag and also depending on whether the protein was expressed individually or simultaneously with other proteins. Finally, we demonstrated that bacteria with surface-displayed evasins and affitins are functional, i.e., capable of binding to IL-8 and the EpCAM receptor. Due to the general disapproval of GMOs, we separated the expressed proteins from the producer cells, attached them non-covalently to wild strains of bacteria that were not genetically modified, and proved their surface presentation. The entire evaluation of proteins was performed on the basis of peptide tags and demonstrated that their use is a suitable, affordable and easy way to detect and differentiate expressed proteins, which was also the basic purpose of the master's thesis. We have found that although peptide tags sterically interfere with the binding sites of proteins and affect their functionality, the advantages of their use outweigh the mentioned disadvantages.

Polovica bolnikov z rakom debelega črevesa in danke (RDČD) bo med boleznijo razvila jetrne zasevke. Pri nezdravljenih jetrnih zasevkih RDČD je prognoza slaba. Za te bolnike je radikalen kirurški poseg edino potencialno kurativno zdravljenje. V zadnjih letih se je preživetje bolnikov z jetrnimi zasevki izboljšalo zaradi številnih novosti v kirurškem zdravljenju in tudi zaradi razvoja številnih novih metod zdravljenja. V nekaterih raziskavah poročajo celo o 60-odstotnem 5-letnem preživetju. Več kot 4 zasevki, zasevek, večji od 5 cm, bilobarna bolezen in zunajjetrni zasevki niso več kontraindikacije za operacijo jetrnih zasevkov, ampak sta za odločitev o operaciji pomembna predvsem dva dejavnika: sposobnost doseči vsaj 1 mm širok varnostni rob ob ohranitvi vsaj 20 do 30 % zdravega jetrnega parenhima. Umrljivost po operaciji jetrnih zasevkov je pri ustreznem izboru bolnikov in dobri predoperativni pripravi manjša od 5 %. Approximately one half of patients with colorectal cancer will develop liver metastasis at some point in their clinical course. Untreated colorectal liver metastases have a bad prognosis and complete surgical resection is the only treatment that offers a chance of long term survival. In recent years, the survival of patients with colorectal liver metastasis has improved due to several advances in surgical techniques and development of other new methods of treatment. In some studies, 5-year survival is approaching 60%. More than four lesions, tumors larger than 5 cm, bilobar disease and extrahepatic disease are no longer considered as contraindications for surgery. Nowadays, the eligibility criteria for surgery are feasibility of R0 resection and making sure that at least 20-30% of the liver will remain intact. With careful selection of patients and proper preoperative care, the mortality rate after liver resection is estimated at less than 5 %.

Rak debelega črevesa in danke (RDČD) je tretja najpogostejša vrsta raka in hkrati četrti najpogostejši vzrok smrti zaradi rakavega obolenja na svetu. Čeprav je izvajanje presejalnih testov za raka debelega črevesa v polnem zagonu, in se je stopnja preživetja tekom zadnjih tridesetih let dvignila, je zaradi pomanjkljive zaznavnosti velike večine testov v zgodnji fazi odkritih zgolj okrog 40 – 44 % rakov. V kolikor je rak diagnosticiran v zgodnjem stadiju (I in II) je preživetje pacientov občutno višje kot pri detekciji v poznejšem stadiju (III in IV), kar pomeni da je zgodnja detekcija RDČD izjemno pomembna in potrebna. Trenutni presejalni testi (predvsem imunokemijski testi blata) imajo nižjo sensitivnost v zgodnjem stadiju ter nizko komplianco pacientov, kar pomeni, da obstaja potreba po izboljšavi presejalnih testov. Sprememba presnove je značilen znak ob pojavu raka, ki se kaže v spremembah signalnih poti. Metaboliti so končni produkti celičnih procesov, njihove koncentracije pa se odražajo v delovanju organizma in so hkrati tesno povezane z izraženim fenotipom, kar pomeni, da so metaboliti primerni biološki označevalci za zaznavanje rakavih bolezni. V sklopu te raziskave je bilo pokazano, da je na podlagi metabolnega profila seruma možno razlikovati med pacienti z RDČD, adenomatoznimi polipi in zdravimi pacienti. Vendar je do same uporabe takšnega testa v klinični praksi potrebno še zaključiti multicentrično klinično validacijo na asimptomatski populaciji. Na podlagi te klinične validacije se določi realna natančnost presejalnega testa in preuči potencialna uvedba testa v zdravstven sistem. Colorectal cancer (CRC) is the third most common type of cancer and the fourth most common cause of cancer death in the world. Although the implementation of creening tests for CRC is in full swing, and the survival rate has risen during the last thirty ears, only about 40-44% of cancers are detected in the early stage, which is due to the limitations of existing tests. If the cancer is diagnosed in the early stage (I or II), the survival of patients is significantly higher compared to detection in later stage (III or IV). This indicates that early detection of CRC is extremely important and necessary. Current screening tests (especially fecal immunochemical test - FIT) have lower sensitivity for early stage CRC and low patient compliance, which means there is a great need to improve existing screening tests. Altered metabolism is one of typical signs of cancer, which can be seen as changes of specific signal pathways. Metabolites are final products of cellular processes, and their concentrations reflect the functioning of organism and are closely related to the phenotypic, which means metabolites are suitable biological markers for the detection of cancers. As part of this study, it has been shown that specific metabolic profile of the serum can be used to distinguish between patients with CRC, adenomatous polyps and healthy patients. However, to use such a test in clinical practice, multicentre clinical validation study on an asymptomatic population has to be completed. Such clinical validation will determine the actual accuracy of the screening test and will show the potential for introduction of the test into the healthcare system.

Background. There is now compelling evidence that screening for colorectal cancer may result in significantly reduced mortality. Screening tests for colorectal cancer are not systematically performed in Bulgaria. Aim. This article explores the effect of an educational intervention on the willingness of patients to participate in the screening for colorectal cancer with the immunochemical faecal occult blood test in the home setting. Materials and methods. A before-after design study of the effects of educational intervention comprising distribution of a brochure and one-to-one discussion with a GP. A self-administered, original questionnaire was administered before and after the intervention to 600 randomly selected patients in 40 general practices (15 patients per practice) in Plovdiv district. Results. The intervention led to an increase with >20% of the patient’s knowledge of the importance of the test and on how to carry out the test. Statistical analysis indicated that there was an increase in knowledge after the educational intervention about the usefulness of the test (24.8% in males, 18.3% in females) and its performance (22.7% in males, 25.4% in females). Conclusion. The educational intervention has significantly influenced the patient’s awareness about the test’s usefulness and its self-administration. It improved the awareness by providing an easy access to information, thus fostering the active involvement of the patients. A strength of the intervention was the patient-centered approach in providing additional information through one-to-one discussions, and it ensured a higher quality of the preventive screening in the general practice.

Bolezen kot je rak debelega črevesa in danke je ena izmed najpogostejših bolezni raka v Sloveniji kot tudi v Evropi. Z namenom, da bi zmanjšali porast te bolezni, so v letu 2008 uvedli preventivni državni Program SVIT. Program zajema žensko in moško populacijo ljudi, staro med 50 in 69 let. Z raziskavo želimo ugotoviti, kakšna je vloga medicinske sestre, da bi povečale odzivnost, zakaj ravnajo tako in kje one vidijo zapreke glede na odzivnost pacientov, ki jih zaznajo pri svojih intervencijah. Prav tako nas je zanimalo ali jih delodajalec podpira in kako poteka sodelovanje med sodelavci Programa SVIT in medicinskimi sestrami. Disease such as colorectal cancer is one of the most common cancers in Slovenia as well as in Europe. In order to reduce the growth of this insidious disease, the national SVIT program was introduced in 2008. The program includes male and female population, aged between 50 and 69 years. The survey is aimed at determining the role of nurses in order to increase responsiveness, why they act so and where they see obstacles considering the responsiveness of the patient, which can be detected in their interventions. Furthermore, we were interested in how much support they have from the employer, and how the interaction with the SVIT program and nurses takes place.