High Frequency of BRAF Proto-oncogene Hot Spot Mutation V600E in Cohort of Colorectal Cancer Patients from Ahvaz City, Southwest Iran
Javad Mohammadi Asl,
Mohammad Amin Tabatabaiefar
Colorectal cancer (CRC) is one of the most common forms of cancer around the world. Sporadic CRCs are caused by accumulation of mutations in essential genes regulating normal proliferation and differentiation of cells. The proto-oncogene BRAF encoded by the BRAF gene is involved in the RAS/RAF/MAPK pathway of signal transduction during cell growth. Acquired mutations in BRAF have been found at high frequencies in adult patients with papillary thyroid carcinoma and sporadic CRC. One of the predominant hot spot point mutations is T1799A (V600E) mutation among a cohort of CRC patients from Ahvaz city, southwest Iran. The aim of this study was to estimate the frequency of V600E mutation in CRC patients from Ahvaz city, southwest Iran. We analyzed exon 15 of the BRAF gene in isolated DNA from 80 Formalin Fixed Paraffin-embedded (FFPE) CRC tumor tissues using PCR-RFLP method. Data were analyzed using SPSS statistical program. According to our results 37 out of 80 cases (46.25%) were heterozygous for the mutation while the remaining 43 cases (53.75%) had normal homozygous genotype. No homozygous mutant genotype was found. Based on our findings, the frequency of V600E mutation appears to be significantly increased among CRC patients of the studied population but there was no significant relationship between genotypes and age and sex. In conclusion, these findings might prove the effect of V600E mutation on CRC pathogenesis. However, the exact effect of the mutation in CRC progression requires further work.
Received: April 12, 2013;
Accepted: May 08, 2013;
Published: November 26, 2013
Colorectal cancer (CRC) is one of the most prevalent types of cancers (Rasuck
et al., 2012). Possibly due to progressive proliferation of cancer
cells in the colon, rectum and vermiform appendix. Adenocarcinoma of colon is
a malignant tumor starting out as benign adenomas whose polyps may gradually
increase in size and develop into cancer. Although CRC can manifest in all ages,
it mostly occurs after age 50. CRC mostly starts from the appearance of small
mushroom-shaped polyps in the bowel wall which are benign but after sometimes
may grow and develop into cancer. Invasive cancers which develop inside the
colon wall (stage I and II) are commonly curable by surgery. Available data
from England and Wales suggest that more than 90% of diagnosed patients in this
stage survive beyond 5 years after diagnosis. If patients are left untreated,
polyps would spread in regional lymph nodes (stage III). As reported in England
and Wale, near 48% of diagnosed patients in this stage continue their life beyond
5 years after diagnosis. Cancers which metastasize to distance region (stage
IV) are not usually curable while approximately 7% of these patients survive
beyond 5 years after diagnosis (Woodman et al.,
2001; Rachet et al., 2009). CRC is more
widespread in developed countries (Australia, New Zealand, Europe and North
America) in compare to its lowest rates in Africa and Asia (Jemal
et al., 2011; Center et al., 2009).
Although resulted data from Tanzanian population revealed that the Colorectal
Cancer was not uncommon in that area (Chalya et al.,
2013). According to Kamangar et al. (2006),
it is reported as the third commonest cancer among women and fourth one among
men (Kamangar et al., 2006; Rachet
et al., 2009). More than half of the CRC victims live in developed
part of the world (Ferlay et al., 2010).
Hereditary Non Polyposis Colorectal Cancer (HNPCC) is the most prevalent type
of familial CRC which accounts for 0.9-2.5% of all CRCs (Kerber
et al., 2005). Germ line mutation in DNA Mismatch Repair (MMR) genes
may lead to HNPCC especially due to mutations in the MLH1 or MSH2 genes. Most
of the cases of this cancer show high levels of DNA microsatellite instability
(MSI) (Aaltonen et al., 1998). Furthermore,
5-10% of CRCs are due to hypermethylation of the MLH1 gene promoter (Kane
et al., 1997). Recently, a sporadic type of CRC has been discovered
in patients who show defect in BRAF protein (Davies et
The BRAF gene encodes the BRAF proto-oncogene which is a Serin/threonine-protein
kinase (Sithanandam et al., 1990, 1992).
The BRAF protein is involved in signal transduction during cell growth. Some
acquired mutations in the gene may lead to adult cancers such as adenocarcinoma
of colon (Davies et al., 2002). Based on some
findings, 90% of BRAF mutations leading to CRC is due to T1799A transversion
in exon 15 leading to V600E (valine replacement by glutamine) (Rajagopalan
et al., 2002; Wang et al., 2003).
As the result, the BRAF proteins structure and function are negatively
affected. Later, the mutation was found to play roles in different adult cancers
such as Papillary Thyroid Cancer (PTC) and about 30-75% of sporadic MSI-H (high
microsatellite instability) CRCs (Wang et al.,
2003; Deng et al., 2004; Kambara
et al., 2004; Vandrovcova et al., 2006).
High prevalence of CRCs in Iranian population as well as other countries encouraged
us to test frequency of the commonest mutation in the BRAF gene. The purpose
of this study was to estimate the V600E mutation frequency in CRC patients and
to determine its association with CRC occurrence. Furthermore, the correlation
between sex and age and occurrence of the acquired point mutation V600E were
MATERIALS AND METHODS
Subjects: Formalin Fixed Paraffin Embedded (FFPE) colorectal adenocarcinoma
tissues were collected from patients filed at Emam khomeini hospital of Ahvaz,
southwest Iran, diagnosed between 2000-2010 during biopsy or surgery. Totally,
80 samples from patients with advanced CRC (metastasis) were included in the
study. The mean age of patients was 44.25 years. Thirty six out of the 80 patients
were female and the remaining patents were male. Most cases regardless of their
gender were in 40-50 age range.
This study was approved by the Review Board of the School of Medicine, Ahvaz
Jundishapur University of Medical Sciences.
Genotyping: FFPE blocks were prepared to 10 μm thick sections by
microtome and collected in 1.5 mL microtubes. The blocks were treated by xylene
to remove the paraffin. Genomic DNA was extracted by QIAamp DNA FFPE Tissue
kit (Cat#56404) (Qiagen, Germany).
PCR-RFLP: Isolated DNA was amplified by PCR using the following pair
of specific primers for exon 15 of the BRAF gene:
||5TCA TGA AGA CCT CAC AGT AAA AAT3 (Forward) and
5TGG ATC CAG ACA ACT GT T CAA3 (Reverse) (Takahashi
et al., 2007). The PCR thermal program was as follows: initial
denaturation at 95°C for 5 min, followed by 40 cycles for denaturation
at 95°C for 30 sec, annealing at 60°C for 30 sec and elongation
at 72°C for 30 sec. After the last cycle, a final extention at 72°C
for 5 min was performed. Amplicons were digested by the restriction enzyme
TspRI (New England Biolabs, Ipswich, MA) at 60°C for 16 h. TspRI recognizes
and cuts the ACAGTGAAA restriction site in the wild type genotype. However,
T1799A point mutation, which replaces T by A, removes the restriction site.
Therefore, the 98 bp PCR product will remain intact. In the presence of
the normal genotype, two distinct 46 and 52 bp fragments are produced. RFLP
products were visualized on 8% Poly Acrylamid Gel Electrophoresis (PAGE)
after ethidium bromide staining. DNA bands were distinguished by use of
100 bp ladder (GeneOn GmbH, Germany).
DNA sequencing: DNA sequencing of the PCR product was carried out bi-directionally
on an ABI 3130 automated sequencer (Applied Biosystems) (Bioneer, South Korea)
using the same primers. The results were using Chromas lite version 2.0 software.
Statistical analysis: The results were represented as the frequency
and percentage of each genotype. Based on results, there were 43 patients with
normal genotype (A/A) which was equal to 53.75%. Additionally, 37 out of 80
patients carried mutant genotype (A/T) which was 46.25% of all CRC tested patients.
We also considered the relationships between genotypes, age and sex. Forty four
and 36 out of the 80 patients were female and male, respectively. 37.5% of cases
were in 40-50 age range (Table 1). Data were analyzed using
SPSS 16.0 computing program and relationships were tested by χ2-test
(Table 2). No significant relationships were observed between
sex or age and BRAF genotypes.
|| Age and sex frequencies in CRC patients
|| Relationships between age or sex and BRAF genotypes
We tested 80 isolated DNA samples from FFPE colorectal cancer (CRC) tissues
to identify the frequency of V600E mutation among Ahvaz CRC patients. Using
the PCR-RFLP leaded to finding mutation in 37 (46.25%) CRC cases. All mutations
were in heterozygous state carrying T→A transversion at nucleotide 1796
identified by sequencing. Forty three samples (53.75%) showed normal homozygous
genotype. The results were represented as frequency and percentage of each genotype
in statistical analysis. As expected, normal homozygous genotypes produced two
bands on PAGE include 47 and 52 bps while heterozygotes showed 3 bands: 47,
52 and 100 bp (Fig. 1). Male and female frequencies and age
ranges are also shown in Table 2. Based on χ2
test results regarding testing correlation between sex (OR = 0.67, df = 1, p
= 0.42) or age (OR = 0.79, df = 1, p = 0.603) and genotypes, no significant
relationships were observed (Table 2). Although most patients
were in 40-50 age range, their ages did not influence their BRAF genotype and
Regarding the high frequency of colorectal cancer (CRC) in Iran and the dramatic
role of BRAF mutation in CRC malignancy and progression, it was necessary to
estimate the frequency of V600E mutation as the most common point mutation in
CRC patients from Ahvaz city, Iran. Due to the present problems in collecting
human samples, we were able to conduct our research with approximately small
number of tumor samples. Totally we have tested 80 CRC samples, among which
46.25% (37 samples out of 80) showed heterozygous mutant genotype. The results
support those of previous studies addressing the BRAF mutations in carcinomas
(Davies et al., 2002; Deng
et al., 2004; Benvenuti et al., 2007).
In the present study, we have obtained the frequency of V600E mutation to be
53.75% in a cohort of CRC patients sampled during 2000-2010. About 91% of CRC
with MSI-H (Jensen et al., 2008) and 15% of
all sporadic CRCs (Deng et al., 2004) are reported
to be positive for BRAF mutations. Thus, our results corroborated previous studies
on BRAF mutation frequencies in CRC patients.
||Position of RFLP bands after cutting by TspRI restriction
enzyme to identify V600E BRAF mutation. From left to right, samples no.
2, 6 and 8 are heterozygous while sample no 4 shows homozygous genotype.
M: Marker, U: Uncut, C: Cut, M: Mutant, N: Normal, bp: Base pair
Although, direct DNA sequencing as a highly sensitive and precise methodology,
was applied to confirm our results, several factors including the relatively
small sample size, presence of the normal tissue beside the malignant one in
FFPE block and presence of most of the tumors in metastasis phase can lead to
the high frequency of the mutation in heterozygous state obtained in this study.
No direct and significant relationships were found in our study between age
and BRAF genotypes. Misdiagnosis of patients in younger ages with BRAF mutation
or poor prognosis of disease might lead to manifestation of complexity encountered
in later ages. Based on recent studies, BRAF mutations lead to poor prognosis
and late diagnosis of the disease (Yokota et al.,
2011). Our results would confirm those of recent studies which showed no
association between BRAF mutations and the patients sex and age (Yuen
et al., 2002).
BRAF genotyping is important for several reasons: different studies have shown
that BRAF mutations can affect the therapeutic response to anti epidermal growth
factor receptor (anti-EGFR) therapy. Cetuximab (Erbitux) and Panitumumab (Vectibix)
are two monoclonal antibodies that inhibit EGFR used in chemotherapy to treat
CRC and PTC patients (Meyerhardt and Mayer, 2005).
Based on Cappuzzo et al. (2008) research, CRC
patients with BRAF mutations had poor response to anti-EGFR therapy (Cappuzzo
et al., 2008; Di Nicolantonio et al., 2008).
According to another study performed in Italy, BRAF V600E mutation may account
for another 12% of resistant cases to anti-EGFR therapy (Benvenuti
et al., 2007). Other investigations carried out in France (Laurent-Puig
et al., 2009), Italy (Loupakis et al.,
2009) and Belgium (De Roock et al., 2010)
support the result. On the other hand, it has been explained that LV5FU-Oxaliplatin
combination seems beneficial as first-line therapy in advanced CRC with BRAF
mutations (De Gramont et al., 2000). Finally,
some of the investigators believe that BRAF mutations especially V600E transition
can be used as a biomarker for selecting patients suitable for anti-EGFR treatment.
The importance of the mutation determination in cancer patients might be proved
by its effect on response to the treatment.
According to our findings, there was a significant relationship between BRAF
genotype and disease occurrence, but no significant correlation between genotype
and patients age and sex were
While there are various involved genes and mutations which may lead to CRC
development and progression, further studies should be performed in the Iranian
population to help finding the most prevalent mutation in the context of genetic
background and environmental factors.
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