Keywords
Colorectal cancer - Iran - KRAS - NRAS
Introduction
Colorectal cancer (CRC) is one of the most common cancers and is the second leading
cause of cancer death in men and women in the United States and also is third common
cancer in women and fifth in Iranian men.[1] The increasing incidence of CRC in the past three decades in Iran has made it as
a major public health burden.[2] In CRC patients, metastases are the main cause of cancer-related mortality.[3] The most common sites of metastasis from colon cancer are the regional lymph nodes,
the liver, the lung, and the peritoneum.[3],[4] Distant metastatic disease is present in approximately 25% of patients at initial
diagnosis, and half of CRC patients will develop metastatic disease.[5] Most patients with metastatic CRC (mCRC) have an incurable disease.[3] The treatment of mCRC is one of the biggest successes in recent decades.[6] Targeted cancer therapy is becoming a powerful strategy for the treatment of patients
selected on the basis of their molecular characteristics. This is particularly true
for patients with mCRC.[2] RAS mutations are useful markers for predicting responses to anti-epidermal growth
factor receptor monoclonal antibodies in mCRC.[2],[7] KRAS mutation varies between 20% and 50% in the most countries in the world,[8] but NRAS mutations are rare and occur in 3% and 5% of CRC.[9] The frequency of NRAS mutations and their relationship to clinical, pathologic,
and molecular features remains uncertain.[9]
The aim of this study is another report from the prevalence of KRAS and NRAS mutations
in Iran and the correlation between KRAS mutation status with clinicopathological
factors and survival.
Materials and Methods
Patients
The cross-sectional study was done in CRC patients in Rasool Akram Hospital, Tehran,
in 1 year (April 2015 to April 2016) that 144 patients were entered into the study
based on inclusion criteria.
Inclusion criteria
Patients having mCRC and history of treatment with chemotherapy regimens were included
in the study. Age, sex, tumor site, grade, metastasis location, familial history,
KRAS/NRAS status, and survival were checked for all patients. The patients were followed
up for 1 year. The overall survival was defined as the length of time from either
the date of diagnosis or the start of treatment for cancer until death for any cause
or the date of the last follow-up.
Extraction and amplification of KRAS/NRAS
Mutations and oncogenes of KRAS and NRAS of codons 12 and 13 were checked on fresh
frozen and formalin-fixed paraffin-embedded (FFPE) tissues in Partolab Laboratory,
Tehran, Iran. After 4–5 cutting with 2–5 μm thickness, DNA was extracted with FFPE
QIAGEN kit, and then, polymerase chain reaction (P CR) for amplification of gene segments was done with an initial denaturation at 95°C
for 11 min, denaturation at 95°C for 30 s, fusion at 55°C for 30 s, elongation at
72°C for 30 s, and end elongation at 72°C for 5 min. After electrophoresis of PCR
products, a band in the situation of 120 bp was seen that is indicating amplification
of gene segment during PCR. To ensure amplified fragment length to confirm the target
gene, evaluation was performed by Gene Runner program, and the incision with enzyme
was confirmed by MapViewer program. After that, using RFLP technic and suitable enzymes
(Bgl1), the status of mutation and wild type was determined. The results again were
checked with high-resolution melting analysis. KRAS and NRAS genes were analyzed and
sequenced (pyrosequencing) by allele specific.
Statistical analysis
The analysis was done with IBM SPSS software version 22 (IBM Corp., Armonk, NY, USA)
that t-test was used for the comparison of means between groups and Chi-square test
for other variables. The overall survival was plotted and analyzed by Kaplan – Meier.
Results
The mean age ± standard deviation at diagnosis was 52.9 ± 12.7 years (range: 27–72
years) that 39.6% patients had age <50 years and 54.2% were males [Table 1]. Sigmoid (33.4%), ascending colon (31.2%), rectum (20.8%), descending colon (8.4%),
and transverse colon (6.2%) were the highest tumor site in the patients. Grades I
(well differentiated), II (moderate differentiated), and III (poorly differentiated)
were 16.7%, 64.6%, and 18.7%, respectively. In all patients, liver metastasis (72.9%)
had the highest prevalence, followed by nonregional lymph node and lung (each 10.4%)
and other metastases (6.3%). Of 144 patients, 15 patients had a familial history of
cancer, 72 (52.1%) had KRAS mutation, and 6 (4.2%) had NRAS mutation. In addition,
during 1-year follow-up, 51 (35.4%) patients died for any cause.
Table 1
Characteristics of all patients (n=144)
|
Variables
|
n (%)
|
|
SD – Standard deviation; KRAS – Kirsten ras; NRAS – Neuroblastoma ras
|
|
Age (years)
|
|
Mean±SD
|
52.9±12.7
|
|
Range
|
27-72
|
|
<50
|
57 (39.6)
|
|
Sex
|
|
Male
|
78 (54.2)
|
|
Female
|
66 (45.8)
|
|
Tumor site
|
|
Ascending colon
|
45 (31.2)
|
|
Transverse colon
|
9 (6.2)
|
|
Descending colon
|
12 (8.4)
|
|
Sigmoid
|
48 (33.4)
|
|
Rectum
|
30 (20.8)
|
|
Grade
|
|
I
|
24 (16.7)
|
|
II
|
93 (64.6)
|
|
III
|
27 (18.7)
|
|
Metastasis location
|
|
Liver
|
105 (72.9)
|
|
Nonregional lymph node
|
15 (10.4)
|
|
Lung
|
15 (10.4)
|
|
Other
|
9 (6.3)
|
|
Familial history
|
|
Yes
|
15 (10.4)
|
|
No
|
129 (89.6)
|
|
KRAS status
|
|
Mutation
|
75 (52.1)
|
|
Wild type
|
69 (47.9)
|
|
NRAS status
|
|
Mutation
|
6 (4.2)
|
|
Wild type
|
138 (95.8)
|
|
One-year survival
|
|
Alive
|
93 (64.6)
|
|
Deceased
|
51 (35.4)
|
[Table 2]compares the characteristics of the patients based on KRAS status. There was just
a significant difference between KRAS mutation patients and KRAS wild-type patients
(P < 0.001) that KRAS mutation was more in the patients with age ≥50 but KRAS wild type
in the patients with age <50 years.
Table 2
The comparison of characteristics of the patients based on KRAS status
|
Variables
|
KRAS mutation (n=75) (%)
|
KRAS wild type (n=69) (%)
|
P
|
|
SD – Standard deviation; KRAS – Kirsten ras
|
|
Age (years)
|
|
Mean±SD
|
54.64±13.11
|
51.09±12.04
|
0.093
|
|
<50/≥50
|
18 (24)/57 (76)
|
39 (56.5)/30 (43.5) <0.001
|
|
Sex
|
0.645
|
|
Male
|
42 (56)
|
36 (52.1)
|
|
Female
|
33 (44)
|
33 (47.9)
|
|
Tumor site
|
0.574
|
|
Ascending colon
|
24 (32)
|
21 (30.4)
|
|
Transverse colon
|
6 (8)
|
3 (4.3)
|
|
Descending colon
|
6 (8)
|
6 (8.7)
|
|
Sigmoid
|
21 (28)
|
27 (39.2)
|
|
Rectum
|
18 (24)
|
12 (17.4)
|
|
Grade
|
0.914
|
|
I
|
12 (16)
|
12 (17.4)
|
|
II
|
48 (64)
|
45 (65.2)
|
|
III
|
15 (20)
|
12 (17.4)
|
|
Metastasis location
|
0.436
|
|
Liver
|
57 (76)
|
48 (69.5)
|
|
Nonregional
|
6 (8)
|
9 (13.1)
|
|
lymph node Lung
|
9 (12)
|
6 (8.7)
|
|
Other
|
3 (4)
|
6 (8.7)
|
|
Familial history
|
0.322
|
|
Yes
|
6 (8)
|
9 (13.1)
|
|
No
|
69(92)
|
60 (86.9)
|
The comparison of 1-year overall survival based on KRAS status has been shown in [Figure 1]. The 6-month overall survival rate in KRAS mutation patients (84%) was more than
KRAS wild-type patients (98.6%) (P = 0.002). Furthermore, the 1-year overall survival rate was 68% in KRAS mutation
patients versus 60.9% in KRAS wild-type patients (P = 0.371).
Figure 1: The comparison of 1-year overall survival based on KRAS status
Discussion
This study showed that the prevalence of KRAS and NRAS mutations in mCRC patients
in an Iranian population was 52.1% and 4.2%, respectively. Furthermore, KRAS mutation
had significantly more prevalence in lower ages compared with KRAS wild type.
One study [10] in Western Iran on 83 mCRC patients (mean age: 57.7 years, range: 18–80 years, and
61.4% males) and another study on 33 mCRC patients [11] (mean age: 51.5 years, range: 22–76, and 79% males) in this area reported that the
prevalence of KRAS mutation was 33.3% and 44.6%, respectively. Furthermore, there
were no significant differences between patients with KRAS mutation and wild type
in 5-(69% vs. 64%)[10] and 2-year (63% vs. 73%) overall survivals.[11] Two studies [10],[11] showed that the prevalence of clinicopathological factors in patients with KRAS
mutation and wild type was similar. Two studies in Tehran (Central Iran)[1],[12] evaluated mCRC patients for KRAS status that the first study selected 1000 cases
(57.3% males) with the prevalence of 33.6% for KRAS mutation, and the second study
selected 182 CRC patients with the prevalence of 37.4% for KRAS mutation. Koochak
et al.[13] indicate that KRAS mutations occurred at a statistically higher frequency in older
patients (>50) than in younger patients (≤50) (P = 0.0001). It is also worth mentioning that KRAS mutation tended to occur at a more
frequency in male cases than in female cases. KRAS mutations occur at a higher rate
in pT3 than others (P = 0.0001). The studies in other areas of Iran reported 32% KRAS mutation in 100
cases (mean age: 59 years and 55% males) in Southern Iran,[14] 32.2% in 211 cases in Northern Iran,[13] and 28% in 50 cases (mean age: 60.8 years), and NRAS mutation was just in one case
in codon 146 (2%) in Southern Iran.[15] The prevalence of NRAS mutation in Iranian CRC patients has been reported from 0%[6],[10],[11] to 4.2% (the present study). One review study [7] showed that the prevalence of KRAS mutation in Iran was between 30% and 50% (few
studies reported 12.5%–37.4%) that this prevalence was similar to studies from other
countries (20%–50%).
A total of 83 mCRC patients evaluated in Saudi Arabia [16] that 42.2% had KRAS mutation, and 51% and 23% of the tumors were from the left hemi-colon
and rectum, respectively, 83% were moderately differentiated, and 86% were invasive
adenocarcinoma. Another study on 300 CRC patients from Saudi Arabia [17] reported 42% KRAS mutation that mutations were associated with advanced stage of
CRC and shorter recurrence-free survival and overall survival.
The studies reported in Turkey [18],[19],[20] checked 50, 172, and 53 mCRC patients. The prevalence of KRAS mutation was 30%,
44%, and 49.05%, respectively. KRAS mutation frequency was significantly higher in
tumors located in the ascending colon,[18] and there was no difference in progression-free survival and overall survival between
KRAS mutation and KRAS wild-type patients.
One hundred and fifty CRC patients (64% male) in Pakistan were assessed for KRAS status
that 13% had the mutation, and this mutation seemed to be significantly associated
with female patients.[21] One research in Egypt [22] analyzed KRAS status on 26 mCRC patients with immunohistochemistry. The results
showed that 42.3% patients were KRAS mutation, and no significant correlation was
found between clinicopathological parameters and KRAS staining results.[22] In Oman,[23] 79 CRC patients were checked that 48.1% had mutation and no relation was noticed
with wild-type or mutant KRAS with recurrence-free survival and overall survival.
In Iraq,[24] fifty CRC patients (mean age: 55.4 years and 54% males) enrolled that 48% had KRAS
mutation, and there were no significant associations of age, gender, tumor location
or histology, grading, staging, or lymphovascular invasion with KRAS mutation status.In
the present study, there was no significant difference between clinicopathological
factors with KRAS mutation status, except for age that KRAS mutation frequency was
higher in age ≥50 years. Furthermore, there was no significant difference between
KRAS mutation status for 1-year overall survival, but 6-month overall survival was
significantly shorter in KRAS mutation compared with KRAS wild type.
Discussion
The mCRC was more prevalent in men than women, and the mean age varied around 50–60
years. The results showed that the present study had the highest prevalence of KRAS
mutation in the Middle East and Pakistan with the lowest prevalence in CRC patients.
The studies reported that survival of the patients was similar between KRAS mutation
and wild type, but in the present study, 6-month survival had a significant difference.
In the future, studies can be considered to the survivals of shorter than 1 year in
mCRC patients. In addition to, in more studies, there was no significant difference
between KRAS mutation status with clinicopathological factors but can be considered
to the role of age, stage, and tumor location in the future studies.
