Značajnost određivanja mutacija gena KRAS i BRAF u bolesnika s karcinomom debelog crijeva

doctoral thesis

• Title (english): Significance of KRAS and BRAF mutation testing in colorectal cancer patients
• Author: Ivana Rako
• Mentor: Jadranka Sertić (mentor); MBZ: 124856
• Committee member: Jerka Dumić (predsjednik povjerenstva)
• Committee member: Jasminka Pavelić (član povjerenstva); MBZ: 79061
• Committee member: Jadranka Sertić (član povjerenstva); MBZ: 124856
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; Zagreb
• Defense date and country: 2012-07-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Uvod. Učestalost i smrtnost od karcinoma debelog crijeva u stalnom je porastu i zato je ova bolest vodeći
zdravstveni problem u svijetu i u Hrvatskoj. Nastaje složenim procesom tijekom kojeg dolazi do uzastopnih
promjena u tumor supresorskim genima, onkogenima i genima za popravak krivo sparenih baza. Karcinom
debelog crijeva može se pojaviti sporadično ili kao nasljedni oblik; u oba slučaja radi se o vrlo heterogenoj
skupini zloćudnih tumora. Geni KRAS i BRAF spadaju u skupinu onkogena. Njihovi produkti posreduju u
prijenosu signala od receptora za epidermalni čimbenik rasta (EGFR) na unutarstanične mitogenom aktivirane
protein kinaze (MAPK), te na taj način sudjeluju u kontroli staničnog ciklusa. Najčešće promjene ovih gena su
točkaste mutacije. Događaju se u dobroćudnoj fazi nastanka karcinoma debelog i završnog crijeva i odgovorne
su za klinički značajan rast tumora. Cilj ovog rada je odrediti učestalost i vrstu mutacija gena KRAS i BRAF u
uzorcima karcinoma debelog i završnog crijeva te ispitati da li postoji povezanost mutacija s patohistološkim
pokazateljima lošije prognoze bolesti. Materijali i metode. U istraživanje je uključeno 113 arhivskih uzoraka
(tkiva fiksirana u formalinu i uklopljena u parafin) karcinoma debelog i završnog crijeva osoba koje boluju od te
bolesti, operiranih u razdoblju od 2009. do 2011. godine u KBC-u Zagreb. Za izdvajanje DNA korišteno je 6-10
rezova debljine 10 mikrometara svakog pojedinog tkiva. Sedam najčešćih točkastih mutacija gena KRAS
(p.G12A, p.G12D, p.G12V, p.G12R, p.G12C, p.G12S, p.G13D) određeno je metodom kvantitativnog PCR u
stvarnom vremenu, a jedna mutacija gena BRAF (p.V600E) metodom PCR u stvarnom vremenu uz analizu
krivulje taljenja. Za svakog bolesnika prikupljeni su podaci o dobi, spolu, veličini tumora, histološkom gradusu
tumora, stadiju po Dukes'u, angioinvaziji i smještaju tumora. Usporedba učestalosti mutacija u uzorcima
podijeljenim po skupinama učinjena je Fisherovim testom, statističkom obradom podataka u programu MedCalc
v9.3.2.0. Rezultati. Mutacije gena KRAS dokazane su u 39,8% uzoraka, a najčešće je dokazana mutacija p.G12V
(42,2% uzoraka). Tranzicije i transverzije bile su podjednako zastupljene. Mutacija p.V600E gena BRAF
dokazana je u 8,8% uzoraka i to isključivo u uzorcima s nemutiranim genom KRAS. Učestalost mutacija gena
BRAF bila je viša u loše diferenciranim tumorima i tumorima klasificiranim u stadij bolesti Dukes'C (p = 0,04).
Sve mutacije gena BRAF dokazane su u karcinomima debelog crijeva (p = 0,01). Zaključak. Učestalost mutacija
gena KRAS i BRAF u populaciji bolesnika s karcinomom debelog i završnog crijeva iz Hrvatske nalazi se unutar
granica prijavljene učestalosti istih mutacija u drugim populacijama svijeta. Ističe se visok udio mutacije p.G12V
koja se povezuje s najlošijom prognozom bolesti, što bi se moglo povezati s kontinuirano visokom smrtnošću od
ove bolesti u Hrvatskoj. Viša učestalost mutacija gena BRAF u tumorima lošije prognoze upućuje na njihovu
ulogu u napredovanju bolesti.

Abstract (english)

Introduction. Incidence and mortality of colorectal cancer are on constant increase and represent one of the
major health problems in the world as well as in Croatia. The development of colon cancer is a multistep process
which involves successive genetic alterations of tumor supressor genes, oncogenes and mismatch repair genes.
Colon cancer can arise as sporadic or familial hereditary colon cancer, representing in both cases a very
heterogeneous group of malignant tumors. KRAS and BRAF genes are oncogenes. Their products mediate and
control cellular responses through mitogen activating protein kinase signal pathway (MAPK), downstream of
epidermal growth factor receptor (EGFR). The most common activating mutations of these oncogenes are point
mutations. They arise early during the development of colorectal cancer and are associated with the clinically
significant tumor growth. The aim of this study was to determine the incidence of KRAS and BRAF gene
mutations in colorectal cancer patients in Croatia and to assess whether they are linked with clinicopathological
features of poor prognosis. Materials and methods. A total of 113 consecutive colorectal cancer patients were
included in this research. They were operated on 2009-2011 period at University Hospital Center Zagreb.
Resected tumor samples were formalin fixed and paraffin embedded and prepared for DNA extraction as four to
six 10-μm sections. Seven common point mutations of KRAS gene (p.G12A, p.G12D, p.G12V, p.G12R, p.G12C,
p.G12S, p.G13D) were identified using quantitative real-time PCR, and the most common mutation of BRAF
gene (p.V600E) was detected using real-time PCR by fluorescence melting curve analysis. Clinical and
pathological data such as age, sex, tumor size, histologic grade, Dukes’stage, angioinvasion and tumor position
were collected for each patient. Differences in clinical and pathological variables between the groups of patients
with and without BRAF and KRAS mutations were analyzed using Fisher exact tests performed using MedCalc
v9.3.2.0. Results. KRAS gene mutations were detected in 39.8% samples, and the most frequent mutation was
p.G12V detected in 42.2% positive samples. Transitions and transversions were equally present. BRAF gene
mutation p.V600E was detected in 8.8% samples, exclusively in tumors without KRAS mutations. Incidence of
BRAF gene mutation was higher in poor differentiated and Dukes'C tumors (p = 0.04). All BRAF gene mutations
were detected in colon cancers (p = 0.01). Conclusion. The incidence of KRAS and BRAF gene mutations in
colorectal cancer patients from Croatia is within commonly accepted limits. High percentage of p.G12V
aggressive mutation can be related to high mortality from colorectal cancer in Croatia. High incidence of BRAF
gene mutations in tumors with poor prognostic markers shows that BRAF mutations play a role in the
progression of this disease.

• Keywords: KRAS; BRAF; točkaste mutacije; karcinom debelog crijeva; karcinom završnog crijeva
• Keywords (english): KRAS; BRAF; point mutations; colorectal cancer
• Language: croatian
• URN:NBN: urn:nbn:hr:163:423924
• Study programme: Title: Pharmacy and biochemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor znanosti (doktor znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-12-01 13:31:05