Invest Clin 66(3): 234 - 240, 2025 https://doi.org/10.54817/IC.v66n3a01

Corresponding author: Tuba Devrim, 8780/1 Sk 18, 35620 Çiğli/Izmir, Türkiye. Tel. +905432022088.

E-mail: tuba.devrim@bakircay.edu.tr

A preliminary investigation

of the association between KRAS, NRAS,

and BRAF mutations and colorectal cancer

in Turkish patients.

Tuba Devrim1, Saniye Sevim Tuncer2 and Gamze Erkılınç1

1Izmir Bakircay University, Çiğli Training and Research Hospital, Department of Medical

Pathology, Izmir, Türkiye.

2 Ministry of Health, Çiğli Training and Research Hospital, Department of Medical

Pathology, Izmir, Türkiye.

Keywords: NRAS; BRAF; KRAS; colorectal cancer.

Abstract. Somatic mutations in the GTPase RAS protein family and the

downstream serine-threonine kinase BRAF are predicted to be key driver muta-

tions in colorectal carcinogenesis by disrupting critical control points in cell

cycle regulation. In our study, we aimed to investigate the relationship between

KRAS, NRAS, and BRAF mutations in colorectal cancer (CRC) samples and cor-

responding clinicopathological data. This retrospective study included 64 CRC

patients who were evaluated for KRAS, NRAS, and BRAF mutations in our de-

partment between 2022 and 2024. The findings were evaluated according to

the age, gender, tumor localization in the colon, and histopathological subtype

of the patients in whom the mutation was detected, and the relationships be-

tween these variables were analyzed using the chi-square test. KRAS mutations

were detected at 29.6%, NRAS mutations at 3.1% and BRAF mutations at 1.6%.

No significant relationship was found between mutation rates and the patients’

age, gender and colon localization. Our study demonstrated that mutations in

KRAS, NRAS, and BRAF were not associated with the age, sex, and tumor loca-

tion of CRC patients. The data presented are preliminary findings, and more

research is needed to evaluate the clinical and pathological impact of these

mutations on colorectal cancer progression and outcomes.

KRAS, NRAS, and BRAF mutations unrelated to clinicopathological features in colorectal cancer 235

Vol. 66(3): 234 - 240, 2025

Una investigación preliminar sobre la asociación

entre mutaciones KRAS, NRAS y BRAF en cáncer colorrectal

en pacientes Turcos.

Invest Clin 2025; 66 (3): 234 – 240

Palabras clave: NRAS; BRAF; KRAS; cáncer colorrectal.

Resumen. Se predice que las mutaciones somáticas en la familia de las

proteínas GTPasa RAS y la serina-treonina quinasa BRAF son mutaciones clave

en la carcinogénesis colorrectal al interrumpir puntos de control críticos en la

regulación del ciclo celular. En este estudio, el objetivo fue evaluar la relación

entre las mutaciones KRAS, NRAS y BRAF en muestras de cáncer colorrectal

(CCR) y datos clinicopatológicos. Este estudio retrospectivo incluyó a 64 pa-

cientes con CCR que fueron evaluados para mutaciones en KRAS, NRAS y BRAF

en nuestro servicio entre 2022-2024. Los hallazgos se evaluaron según la edad,

el sexo, la localización del tumor en el colon y el subtipo histopatológico de los

pacientes en los que se detectó la mutación, y se analizaron las relaciones entre

ellos mediante la prueba de chi-cuadrado. Las mutaciones en KRAS se detecta-

ron en un 29,6%, en NRAS en un 3,1% y en BRAF en un 1,6%. No se encontró

una relación significativa entre las tasas de mutación y la edad, el sexo y la ubi-

cación del colon de los pacientes. Nuestro estudio demostró que las mutaciones

en KRAS, NRAS y BRAF no se asociaron con la edad, el sexo y la localización del

tumor de los pacientes con CCR. Los datos presentados son nuestros hallazgos

preliminares y se necesita más investigación para evaluar el impacto clínico

y patológico de estas mutaciones en la progresión y los resultados del cáncer

colorrectal.

Received: 16-01-2025 Accepted: 21-06-2025

INTRODUCTION

The incidence rates of colorectal cancer

(CRC) in adults (under 55 years of age) have

been increasing by 1–2% annually. In the late

1990s, CRC was the fourth leading cause of

cancer death in both men and women un-

der 50. However, it has since escalated to

become the leading cause of cancer death in

men and the second leading cause in women

within this age group 1. CRCs are the second

leading cause of death from malignancy, and

more than half of CRCs become metastatic

2. Despite the substantial impact this disease

has on both quality of life and the healthcare

system, data regarding the molecular analy-

sis of biomarkers in patients diagnosed with

CRC is limited 3.

Several oncogenes, particularly muta-

tions in RAS and BRAF, have a significant in-

fluence on colorectal carcinogenesis. These

mutations lead to the activation of the mi-

togen-activated protein kinase signalling

pathway, which is crucial for cell prolifera-

tion, differentiation, and survival. Activation

of this pathway promotes tumorigenesis by

driving uncontrolled cell growth and facili-

tating processes such as angiogenesis and

metastasis. Understanding the role of these

oncogenes provides insight into the mecha-

236 Devrim et al.

Investigación Clínica 66(3): 2025

nisms of colorectal cancer and identifies po-

tential therapeutic targets 4.

Identifying abnormalities in the KRAS,

NRAS, and BRAF genes is crucial for accu-

rately qualifying CRC patients for treat-

ment with anti-epidermal growth factor

receptor (EGFR) monoclonal antibodies 5.

Oncogenic RAS mutations are found in ap-

proximately 50–55% of metastatic colorectal

cancer (CRC) cases, with regional variations

in prevalence. Importantly, these muta-

tions serve as negative predictive markers

for response to monoclonal antibodies that

target the epidermal growth factor recep-

tor (EGFR). This means that patients with

RAS mutations are less likely to benefit from

EGFR-targeted therapies, making it crucial

to screen for these mutations when deter-

mining treatment options for metastatic

colorectal cancer (mCRC). Identifying RAS

status can help guide more effective and

personalized treatment strategies 4. BRAF

mutations occur in approximately 5–17%

of CRC cases, with the BRAFV600E muta-

tion being the most prevalent. In mCRC, the

existence of the BRAFV600E mutation not

only serves as a negative predictive marker

for response to EGFR-targeted MoAbs but is

also associated with a markedly poor prog-

nosis. This mutation often indicates a more

aggressive disease course and resistance to

conventional therapies, highlighting the

need for alternative treatment strategies

and close monitoring of affected patients.

Understanding the implications of BRAF

status is crucial for optimizing management

and enhancing outcomes in mCRC. The sta-

tus of tumor localization and CC laterality

in determining prognosis and guiding treat-

ment remains controversial. It is thought to

be due to differences in histological, genetic

and immunological features between the left

and right colon 6,7. In the present study, we

hypothesized that somatic mutations in the

GTPase RAS protein family and the down-

stream serine-threonine kinase BRAF could

interfere with essential checkpoints in cell

cycle regulation, acting as significant driv-

ing mutations in colorectal carcinogenesis.

Our goal was to assess the presence of KRAS,

NRAS, and BRAF mutations in clinicopatho-

logical features of CRC samples in our hos-

pital.

PATIENTS AND METHODS

Study population and design

For this retrospective study, KRAS,

NRAS, and BRAF mutations were analyzed

in 64 CRC biospecimens at the Bakırçay

University Faculty of Medicine Laboratory.

Patients were characterized by age, gender,

and tumor location, with rectal and sigmoid

cancers being the most prevalent. The histo-

logical classification of tumors by the WHO

was used as a guide to assign the histologi-

cal subtypes (classical adenocarcinoma and

mucinous adenocarcinoma) and tumor loca-

tion (colon segment other than rectum, and

rectum) 8.

To conduct the study, ethics commit-

tee approval was obtained from the zmir

Bakırçay University Non-Interventional

Clinical Research Ethics Committee on

03.04.2024, with decision number 1681 and

research number 1661.

DNA extraction was conducted using

the QIAamp formalin-fixed paraffin-embed-

ded (FFPE) kit (Qiagen, USA). Tissue sam-

ples were collected at the time of diagnosis

for colon and rectal cancer. FFPE primary tu-

mor samples from 64 CRC patients between

January 2022 and December 2024 were ret-

rospectively reviewed. Mutation analysis was

performed with the KRAS/BRAF, NRAS, and

BRAF Mutation Analysis Kit for Real-Time

PCR (Diatech Easy, Italy). The tests targeted

the most common mutations in exon 2 (co-

dons 12 and 13), exon 3 (codons 59 and 61),

exon 4 (codons 117 and 146) of the KRAS

and NRAS genes, and exon 15 (V600E) of the

BRAF gene.

Statistical Analysis

For the statistical analysis, a chi-square

test was used to examine the associations

KRAS, NRAS, and BRAF mutations unrelated to clinicopathological features in colorectal cancer 237

Vol. 66(3): 234 - 240, 2025

between mutated genes (KRAS, NRAS, and

BRAF) and various clinicopathological vari-

ables, including age, gender, and the specific

localization of the tumor in the colon. The

analysis was conducted using SPSS version

23.0, and p-values of ≤ 0.05 were deemed

statistically significant, contributing to a

better understanding of their implications

in colorectal cancer.

RESULTS

The study included patients aged 33

to 79 years, with a mean age of 62.54 and

a median age of 64. Among them, 41 (64%)

were male and 23 (36%) were female (Table

1). In our study, no mutations were found in

20 patients (31.2%). KRAS exon 2 mutation

was observed in 29.7% of cases, while other

RAS mutations were found in 3.1% of cases,

and the BRAFV600E mutation was seen in

1.6%. One patient had KRAS mutations at

positions 12 and 13, and the other had KRAS

mutations at position 61 and NRAS muta-

tions at position 12.

No associations were found between

the age and gender of CRC patients and the

frequency of KRAS, NRAS, and BRAF gene

mutations. Notably, these mutations were

more frequently diagnosed in men (64%)

than in women (36%). Table 1 shows the dis-

tribution of mutation presence in patients

with colorectal cancer according to age and

gender.

A total of 47 cases had tumor localiza-

tion identified. In 14 cases, the tumors were

located in the left colon, while 33 cases were

localized in the right colon. In the case of

the BRAFV600 mutation, tumor localization

could not be determined. The distribution of

RAS and BRAF mutation values in colorectal

cancer patients according to age, sex and lo-

calization is shown in Tables 2, 3, and 4.

DISCUSSION

Colorectal cancer is the third most

common type of cancer in both sexes in

Turkey. According to the 2024 official

cancer statistics, 152,810 (7.6%) of the

2,001,140 cancer cases diagnosed in 2019

were CRC. Reports indicate a significantly

higher incidence in men (53.3%) com-

pared to women 1,4. In our study, most of

the patients were male (64.1%), consis-

tent with national cancer statistics 1.

Our study aimed to evaluate the pres-

ence of somatic mutations in the GTPase

RAS family of proteins (KRAS and NRAS)

and the downstream serine-threonine kinase

BRAF in CRC samples. It is hypothesized

Table 1. Mutation profile of colorectal cancer

patients stratified by age and gender.

Wild type

n = 44

Mutant

n = 20 p

Age <64 26 (76.5%) 8 (23.5%)

0.125Age ≥64 18 (60%) 12 (40%)

Female 18 (78.3%) 5 (21.7%)

0.172Male 26 (63.4%) 15 (36.6%)

Table 2. Distribution of KRAS, NRAS and BRAF mutations by age in colorectal cancer patients.

KRAS NRAS BRAF

Wild type

n=45

Mutant

n=18

Total

n=63

Wild type

n=62

Mutant

n=2

Total

n=64

Wild type

n=63

Mutant

n=1

Total

n=64

Age <64 24

(38%)

(12.7%)

(50.7%)

(51.6%)

(1.6%)

(53.2%)

(53.1%)

(0%)

(53.1%)

Age ≥64 21

(33.3%)

(15.9%)

(49.2%)

(45.3%)

(1.6%)

(46.9%)

(45.3%)

(1.6%)

(46.9%)

p0.360 0.722 0.469

238 Devrim et al.

Investigación Clínica 66(3): 2025

that these mutations might disrupt criti-

cal checkpoints in cell cycle regulation and

serve as key driving factors in colorectal car-

cinogenesis. The detection rates of KRAS,

NRAS, and BRAF mutations in our cohort

of 64 CRC patients were 29.7%, 3.1%, and

1.6%, respectively. Consistent with the find-

ings of Mosaferi et al. 8, our study also ob-

served that the majority of KRAS mutations

were located in exon 2, while most NRAS mu-

tations were found in exon 3 8. In line with

the findings of Khoshnoudi et al. 2 and Mo-

saferi et al. 8, our study found no significant

association between KRAS mutation status

and age or gender.

The prevalence of KRAS mutations in

our study aligns with findings from the liter-

ature, which consistently reports that KRAS

mutations occur in approximately 40% of

CRC cases 9. These mutations are critical

in terms of poor prognosis and resistance

to targeted therapies, such as anti-EGFR

agents. 10,11 The relatively lower frequency

of NRAS (3.1%) and BRAF mutations (1.6%)

observed in our study is consistent with their

reported rates in the literature, where BRAF

mutations are often associated with more

advanced disease stages and poorer out-

comes 12,13. Our findings showed that muta-

tion rates were not significantly associated

with the patients’ age, gender, or colon lo-

calization.

A study by Yamauchi et al. 14 showed that

the frequency of BRAF mutations increased

from the rectum to the ascending colon and

decreased towards the cecum. Ekmekciu et

al. 15 reported that KRAS mutations were pre-

dominantly located in the right colon, but no

significant differences were observed in age,

stage, or histopathological subtype. Bylsma

et al. 16 found BRAF and RAS mutations more

frequently in right-sided colon cancers, con-

sistent with Ekmekciu et al. 15. In addition,

Bylsma et al. reported that BRAF and RAS

mutations were more common in young pa-

tients 16. Our findings showed that mutation

rates were not significantly associated with

patients’ age, gender, or colon localization.

Table 3. Sex-based frequency of BRAF, KRAS, and NRAS mutations in colorectal cancer patients.

BRAF KRAS NRAS

Wild type

n=63

Mutant

n=1

TOTAL

n=64

Wild type

n=46

Mutant

n=18

Total

n=64

Wild type

n=62

Mutant

n=2

Total

n=64

Female 23

(36%)

(0%)

(36%)

(51.5%)

(6.2%)

(35.9%)

(34.2%)

(1.7%)

(35.9%)

Male 40

(62.5%)

(1.5%)

(64%)

(42.2%)

(21.9%)

(64%)

(62.5%)

(1.5%)

(64%)

p0.414 0.126 0.641

Table 4. Distribution of KRAS and NRAS mutation frequencies according to tumor

location in colorectal cancer.

KRAS NRAS

Tumor Location Wild type

n=31

Mutant

n=16

Wild type

n=45

Mutant

n=2

Total

n=47

Colon 11

(23.4%)

(6.4%)

(19.4%)

(1.5%)

(20.9%)

Rectum 20

(29.9%)

(19.4%)

(47.8%)

(1.5%)

(49.3%)

KRAS, NRAS, and BRAF mutations unrelated to clinicopathological features in colorectal cancer 239

Vol. 66(3): 234 - 240, 2025

We would like to emphasize that the

present study has limitations related to the

cohort size. The limitations of the study in-

cluded that some cases in the study group

were obtained from consultations with exter-

nal centers, while others were obtained from

colonoscopic biopsy materials. For these

reasons, the relationship between prognos-

tic markers for tumor staging and mutation

results could not be compared in our study.

This study is essentially an exploratory, pre-

liminary study, which requires definitive,

confirmatory values. Our findings highlight

the complex interaction between molecu-

lar subtypes, clinical and histopathological

features in CRC and the need for further in-

vestigation of the underlying mechanisms

driving these relationships. In the future,

stratifying patients according to molecular

subtypes may provide advantages in person-

alized treatment approaches.

Acknowledgments

None.

Funding

None.

Conflict of interest

The authors declare that there is no con-

flict of interest associated with this article.

ORCID ID of the authors

• Tuba Devrim (TD):

0000-0002-5321-2002

• Saniye Sevim Tuncer (SST):

0000-0003-0872-7493

• Gamze Erkılınç (GE):

0000-0003-4704-7415

Author contributions

TD and SST designed the study. TD

analyzed and interpreted data and searched

the literature. TD and GE drafted the manu-

script. All authors reviewed the manuscript

and approved the final version.

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