Povezanost promjenljivih faktora rizika i polimorfizama TP53, APC i KRAS gena sa nastankom kolorektalnog karcinoma/

Abstract

Doktorska disertacija se bavi odnosom između promjenljivih faktora rizika i polimorfizama TP53, APC i KRAS gena, sa razvojem kolorektalnog karcinoma i predstavlja važno istraživanje u oblasti kolorektalnog karcinoma (CRC). Ispitana je povezanost faktora rizika za pojavu CRC-a koji se mogu spriječiti i na koje se može utjecati, polimorfizmi TP53, APC i KRAS gena. Varijabilni faktori rizika, koji se mogu spriječiti, su: neumjerena dijeta, gojaznost, dijabetes, fizička neaktivnost, alkohol i pušenje. Studije su pokazale da se mutacije u genima TP53, APC i KRAS često javljaju kod pacijenata sa CRC i dovode do maligne transformacije ćelija. DNK polimorfizmi su varijacije nasljedne osnove (nukleotidna raznolikost) a definiše se kao odnos broja različitih baza i ukupnog broja baznih parova genoma koji se upoređuje. Iako je poznato da se polimorfizmi onkogena i tumor supresorskih gena ( TP53, APC i KRAS geni) često javljaju kod pacijenata sa CRC, nije poznato koliko faktora rizika predstavlja svaki od njih pojedinačno, a koliko njihove kombinacije u pojavi CRC, kao i korelacija polimorfizama ovih gena sa drugim faktorima povezanim sa pojavom CRC. Rezultati ove doktorske disertacije rješavaju molekularne mehanizme kojima polimorfizam gena utiče na težinu, odnosno stadij kolorektalnog karcinoma. Također, identificirani varijabilni i nepromjenljivi (genetički) faktori rizika pomažu nam kao prognostički faktor kod pacijenata sa ovom bolešću i dijagnostički faktor u ranom otkrivanju kolorektalnog karcinoma. Identificiranjem genetičkih faktora povezanih sa pojavom CRC-a i njihovim kombiniranjem sa promjenljivim faktorima, dobijeni su podaci o tome koji su pacijenti najviše izloženi riziku za CRC. Na osnovu identificiranih značajnih faktora povezanih sa pojavom CRC-a, uspostavljeni su obrasci koji bi mogli pomoći u izolaciji ljudi sa povećanim rizikom od CRC-a, što bi moglo omogućiti implementaciju adekvatnih preventivnih mjera na rizičnu populaciju. Istraživanje je sprovedeno prema vrsti istraživačke prospektivne studije slučaja i kontrola u Univerzitetskoj bolnici Foča, Republika Srpska, Bosna i Hercegovina i na Institutu za onkologiju Vojvodine Sremska Kamenica, Srbija tokom 2019. i 2020. godine. Ispitanici su podijeljeni u grupu od 100 slučajeva (CRC pacijenata) i grupu od 100 zdravih kontrolnih pacijenata. Istraživanje je koristilo socio-demografski upitnik, kao i posebno dizajniran upitnik koji je ispitivao prehrambene navike i način života ispitanika, s posebnim naglaskom na vrste hrane, količine i učestalost njihove konzumacije u posljednjih 10 godina. Rutinske laboratorijske analize (kompletna krvna slika) i opći biohemijski nalazi su obavljeni kod svih ispitanika, kao i tumorski biomarkeri CEA i CA 19-9 koji su obavljeni u obje grupe ispitanika. Također, analizirane su endoskopske (lokalizacija tumora) i histopatološke karakteristike tumora (histološki tip tumora, veličina tumora, TNM stadij bolesti, dubina tumorske invazije, histološki stepen, prisustvo metastatskih promjena, broj metastaza i njihova lokalizacija, prisustvo polipa, njihova lokalizacija, veličina, histologija i broj polipa). Dok su uobičajeni aleli jednonukleotidnog polimorfizma (SNP) za gen TP53 varijanta Arg72Pro (rs1042522), APC gen rs459552 i KRAS gen rs8720 varijanta su pojačani tehnikom lančane reakcije polimeraze (PCR). Rezultati studije su pokazali da su pacijenti sa CRC imali značajno veću vjerovatnoću da budu nosioci TP53 (Arg72Pro) Pro/Pro genotipa u odnosu na kontrolne subjekte, a nosioci TP53 Pro/Pro genotipa su imali 2,8 puta veću vjerovatnoću da razviju CRC u odnosu na referentni genotip, dok polimorfizam APC i KRAS gena nije pokazao da su značajni faktori rizika za razvoj CRC. Pacijenti sa CRC su imali značajno veću vjerovatnoću da budu nosioci TP53 Pro / Pro + APC A/T + KRAS C/T kombinacije genotipova u poređenju sa kontrolnim subjektima i imali su 2,6 puta veću vjerovatnoću da razviju CRC u odnosu na referentnu kombinaciju genotipa. Također se dokazalo da je kombinirani genotip TP53 Pro/Pro + APC A/T + KRAS C/T bio značajno više prisutan kod pacijenata sa metastatskim promjenama u odnosu na pacijente bez metastatskih promjena. Pacijenti sa CRC-om su češće imali CRC u porodici, bili su pod stresom, imali su viši nivo stresa, rjeđa fizička aktivnost, pušači su bili češći, češće su konzumirali crveno meso, mesne proizvode, škrobnu hranu i začinjenu hranu, a konzumirali su ribu i zeleno povrće znatno rjeđe u usporedbi s ispitanicima iz kontrolne grupe. Češća konzumacija crvenog mesa i mesnih prerađevina, kao i rijetka konzumacija zelenog povrća, identificirani su kao značajni faktori rizika povezani sa pojavom CRC. U 40% pacijenata sa CRC-om, tumor je lokaliziran u rektumu, u 32% u lijevom debelom crijevu, a 28% u desnom debelom crijevu. U 74% slučajeva, histološki tip tumora bio je adenokarcinom. Bilo je 30% pacijenata u TNM stadiju I i II, 48% u stadiju III i 22% u stadiju IV. Tumor je bio slabo diferenciran u 20%, 48% umjereno i dobro diferenciran u 32% pacijenata. Metastatske promjene u udaljenim organima imalo je 60% pacijenata, dok je prisustvo polipa identificirano u 40%, od kojih je 55% bilo veće od 1 cm. Pacijenti sa CRC imali su značajno veće prosječne vrijednosti neutrofila, CRP, AST, LDH i CK, glukoze u krvi, CEA i CA 19-9 u odnosu na kontrolnu grupu. Pacijenti sa CRC koji su bili nosioci TP53 Pro/Pro genotipa imali su 2,8 puta veće šanse da razviju CRC, dok se polimorfizam APC i KRAS gena nije pokazao kao značajan faktor rizika za razvoj CRC kao pojedinačnih značajnih faktora rizika. Pacijenti sa CRC nosioci kombinacije TP53 Pro/Pro + APC A/T + KRAS C/T genotipovi su imali 2,6 puta veću vjerovatnoću da razviju CRC od referentne kombinacije genotipova. Heterozigotni Pro/Arg genotip gena TP53 bio je značajno češći kod pacijenata bez stresa u odnosu na pacijente koji su bili pod stresom. Homozigotni A/A genotip APC gena bio je češći kod starijih osoba u odnosu na mlađe pacijente, dok nije uočena povezanost kombiniranih genotipova sa nepromjenjivim i promjenjivim faktorima rizika za razvoj CRC. Heterozigotni C/T genotip gena KRAS bio je značajno češći kod pacijenata sa hroničnim bolestima i kod pacijenata sa adenokarcinomom u poređenju sa pacijentima sa mucinoznim adenokarcinomom. Nije uočena značajna razlika u učestalosti različitih pojedinačnih i kombiniranih genotipova TP53, APC i KRAS gena prema Dukes klasifikaciji, lokalizaciji tumora, veličini tumora, TNM stadiju bolesti i histološkom stupnju tumora. Heterozigotni Pro/Arg genotip TP53 gena bio je značajno češći kod pacijenata sa metastazama u odnosu na pacijente bez metastaza. Također, heterozigotni genotip KRAS C/T bio je značajno češći kod pacijenata sa metastazama u odnosu na pacijente bez metastaza. Kombinirani genotip TP53 Pro/Pro + APC A/T + KRAS C/T je bio značajno češći kod pacijenata sa metastatskim promjenama u odnosu na pacijente bez metastatskih promjena.

The doctoral dissertation deals with the relationship between modifiable risk factors and polymorphisms of TP53, APC and KRAS genes with the development of colorectal cancer and represents an important research in the field of colorectal cancer (CRC). The association of preventable and influencable risk factors for CRC, TP53, APC and KRAS gene polymorphisms, was investigated. Variable risk factors, which can be prevented, are: immoderate diet, obesity, diabetes, physical inactivity, alcohol and smoking. Studies have shown that mutations in the TP53, APC, and KRAS genes often occur in patients with CRC and lead to malignant cell transformation. DNA polymorphisms are variations of the hereditary basis (nucleotide diversity) and are defined as the ratio of the number of different bases to the total number of base pairs of the genome being compared. Although it is known that polymorphisms of oncogenes and tumor suppressor genes ( TP53, APC and KRAS genes) often occur in patients with CRC, it is not known how many risk factors each of them individually represent, and how many of their combinations in the occurrence of CRC, as well as the correlation of polymorphisms of these genes with other factors associated with the occurrence of CRC. The results of this doctoral dissertation address the molecular mechanisms by which gene polymorphism affects the severity or stage of colorectal cancer. Also, the identified variable and non-modifiable (genetic) risk factors help us as a prognostic factor in patients with this disease and a diagnostic factor in the early detection of colorectal cancer. By identifying genetic factors associated with the occurrence of CRC and combining them with modifiable factors, data were obtained on which patients are most at risk for CRC. Based on the identified significant factors associated with the occurrence of CRC, patterns have been established that could help isolate people at increased risk of CRC, which could enable the implementation of adequate preventive measures on at-risk populations. The research was conducted according to the type of exploratory prospective case study and controls at the University Hospital Foca, Republic of Srpska, Bosnia and Herzegovina and at the Institute of Oncology of Vojvodina Sremska Kamenica, Serbia during 2019 and 2020. Subjects were divided into a group of 100 cases (CRC patients) and a group of 100 healthy control patients. The research used a socio-demographic questionnaire, as well as a specially designed questionnaire that examined the dietary habits and lifestyle of the respondents, with a special emphasis on the types of food, quantities and frequency of their consumption in the last 10 years. Routine laboratory analyses (complete blood count) and general biochemical findings were performed in all subjects, and tumor biomarkers CEA and CA 19-9 were performed in both groups of subjects. Also, endoscopic (tumor localization) and histopathological characteristics of the tumor (histological tumor type, tumor size, TNM stage of the disease, depth of tumor invasion, histological degree, presence of metastatic changes, number of metastases and their localization, presence of polyps, their localization, size, histology and number of polyps) were analyzed. While the common alleles of the single nucleotide polymorphism (SNP) for the TP53 gene are the Arg72Pro variant (rs1042522), the APC gene rs459552 and the KRAS gene rs8720 variant are enhanced by the polymerase chain reaction (PCR) technique. The results of the study showed that patients with CRC were significantly more likely to be carriers of the TP53 (Arg72Pro) Pro/Pro genotype compared to control subjects, and carriers of the TP53 Pro/Pro genotype were 2.8 times more likely to develop CRC compared to the reference genotype, while the polymorphism of the APC and KRAS genes did not show that they were significant risk factors for the development of CRC. Patients with CRC were significantly more likely to be carriers of the TP53 Pro/Pro + APC A/T + KRAS C/T genotype combination compared to control subjects and were 2.6 times more likely to develop CRC compared to the reference genotype combination. It was also proven that the combined genotype TP53 Pro/Pro + APC A/T + KRAS C/T was significantly more present in patients with metastatic changes compared to patients without metastatic changes. Patients with CRC were more likely to have CRC in the family, were stressed, had higher levels of stress, less physical activity, smokers were more frequent, consumed red meat, meat products, starchy foods and spicy foods more often, and consumed fish and green vegetables significantly less frequently compared to subjects from the control group. More frequent consumption of red meat and meat products, as well as infrequent consumption of green vegetables, have been identified as significant risk factors associated with the occurrence of CRC. In 40% of patients with CRC, the tumor is localized in the rectum, in 32% in the left colon, and 28% in the right colon. In 74% of cases, the histological type of tumor was adenocarcinoma. There were 30% of patients in TNM stage I and II, 48% in stage III and 22% in stage IV. The tumor was poorly differentiated in 20%, 48% moderately and well differentiated in 32% of patients. Metastatic changes in distant organs had 60% of patients, while the presence of polyps was identified in 40% of patients. Patients with CRC had significantly higher mean values of neutrophils, CRP, AST, LDH and CK, blood glucose, CEA and CA 19-9 compared to the control group. Patients with CRC who were carriers of the TP53 Pro/Pro genotype were 2.8 times more likely to develop CRC, while the polymorphism of the APC and KRAS genes has not been shown to be a significant risk factor for the development of CRC as individual significant risk factors. Patients with KRK carriers of TP53 Pro/Pro + APC A/T + KRAS C/T genotypes were 2.6 times more likely to develop CRC than the reference combination of genotypes. The heterozygous Pro/Arg genotype of the TP53 gene was significantly more common in patients without stress compared to patients who were stressed. The homozygous A/A genotype of the APC gene was more common in the elderly compared to younger patients, while the association of combined genotypes with non-modifiable and modifiable risk factors for the development of CRC was not observed. The heterozygous C/T genotype of the KRAS gene was significantly more common in patients with chronic diseases and in patients with adenocarcinoma compared to patients with mucinous adenocarcinoma. No significant difference was observed in the frequency of different single and combined genotypes of TP53, APC and KRAS genes according to the Dukes classification, tumor localization, tumor size, TNM stage of the disease and histological grade of the tumor. The heterozygous Pro/Arg genotype of the TP53 gene was significantly more common in patients with metastases compared to patients without metastases. Also, the heterozygous genotype KRAS C/T was significantly more common in patients with metastases compared to patients without metastases. The combined genotype TP53 Pro/Pro + APC A/T + KRAS C/T was significantly more common in patients with metastatic changes compared to patients without metastatic changes.